Seneca valley virus based compositions and methods for treating disease

ABSTRACT

The present invention relates to a novel RNA picornavirus that is called Seneca Valley virus (“SVV”). The invention provides isolated SVV nucleic acids and proteins encoded by these nucleic acids. Further, the invention provides antibodies that are raised against the SVV proteins. Because SVV has the ability to selectively kill some types of tumors, the invention provides methods of using SVV and SVV polypeptides to treat cancer. Because SVV specifically targets certain tumors, the invention provides methods of using SVV nucleic acids and proteins to detect cancer. Additionally, due to the information provided by the tumor-specific mechanisms of SVV, the invention provides methods of making new oncolytic virus derivatives and of altering viruses to have tumor-specific tropisms.

This application is a continuation of U.S. Ser. No. 13/209,124, whichwas filed on Aug. 12, 2011, which is a continuation of U.S. Ser. No.12/576,296, which was filed on Oct. 9, 2009, now U.S. Pat. No. 8,039,606issued on Oct. 18, 2011, which is a continuation of U.S. Ser. No.11/335,891, which was filed on Jan. 19, 2006, now U.S. Pat. No.7,638,318 issued on Dec. 29, 2009, which is a continuation-in-part ofInternational Application No. PCT/US2004/031504 (InternationalPublication No. WO 2005/030139), which was filed on Sep. 23, 2004, whichclaims priority to U.S. Ser. No. 60/506,182, which was filed on Sep. 26,2003. This application also claims priority to U.S. Ser. No. 60/664,442,which was filed on Mar. 23, 2005 and U.S. Ser. No. 60/726,313, which wasfiled on Oct. 13, 2005. These applications are hereby incorporated byreference in their entirety.

This disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosure,as it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

All patent applications, published patent applications, issued andgranted patents, texts, and literature references cited in thisspecification are hereby incorporated herein by reference in theirentirety to more fully describe the state of the art to which thepresent invention pertains.

BACKGROUND OF THE INVENTION

Virotherapy holds great promise for treating cancer. Oncolytic viruses,which aim to specifically infect and kill cancer cells, whether nativeand/or engineered, may be more efficacious and less toxic thanalternative treatments, such as chemotherapy and radiation. In addition,oncolytic virus therapy that uses replication competent viruses is theonly therapy known that can amplify the therapeutic at thepharmacologically desired site.

A key aspect of cancer therapy is to achieve a high rate of killing ofcancer cells versus normal cells. Accomplishing this goal has beendifficult for many reasons, including the wide array of cell typesinvolved, the systemic dissemination of cancer cells due to metastases,and the narrow biological differences between normal and cancer cells.While progress has been made, much still needs to be done to improveupon current cancer therapies.

In the past, surgeons have tried to remove tumors surgically withoutsubstantially harming the patient. Even complete removal of a primarytumor does not ensure survival since earlier metastases to unknown sitesin the body are left undetected. There is also some research suggestingthat surgical intervention may enhance the growth of distant metastasesdue to removal of tumor cells producing angiogenesis inhibitors.Finally, in many cases, the tumor grows back at the original site aftersurgical removal. Radiation aims to selectively destroy the most rapidlyproliferating cells at the expense of the others. However, tumor cellscan escape radiation therapy either by becoming resistant or by being ina non-dividing state during treatment. In addition, radiation is notalways selective in that many normal cells are actively dividing andkilled by the treatment (cells in bone marrow, gastrointestinal cells,hair follicles, etc.).

Like radiation, chemotherapy is not completely selective and thusdestroys many normal cells, and does not kill all tumor cells due todrug resistance and/or division state of the cell. Thus, chemotherapyand radiation therapies exploit a small differential sensitivity thatexists between normal and cancer cells, giving them a narrow therapeuticindex. A small therapeutic index is clearly an undesirable property ofany modality to treat cancer. Therefore, novel cancer therapeuticapproaches overcoming these limitations are desired.

One such novel approach is oncolytic virus therapy. Initially,replication-defective viruses carrying cytotoxic transgenes wereutilized in attempts to treat cancer. However, they were found to beinefficient in transduction of tumors, inadequate spread within thetumor mass and not adequately selective toward cancers. To overcome thislimitation, viruses were either modified to replicate selectively intumor cells or viruses were discovered to have natural tumor-selectiveproperties. These oncolytic viruses thus had the properties toreplicate, spread, and kill tumor cells selectively through a tumor massby locally injecting the virus or by systemically delivering the virus(FIG. 1).

Despite the early promise of this newly defined class of anti-cancertherapeutics, several limitations remain that may limit their use as acancer therapeutic. Therefore, there is an ongoing need for noveloncolytic viruses that can be utilized for cancer therapy.

SUMMARY OF THE INVENTION

A novel RNA picornavirus has been discovered (hereafter referred to asSeneca Valley virus (“SVV”)) whose native properties include the abilityto selectively kill some types of tumors. As demonstrated below in theexamples, SVV selectively kills tumor lines with neurotropic properties,in most cases with a greater than 10,000 fold difference in the amountof virus necessary to kill 50% of tumor cells versus normal cells (i.e.,the EC₅₀ value). This result also translates in vivo, where tumorexplants in mice are selectively eliminated. Further, in vivo resultsindicate that SVV is not toxic to normal cells, in that up to 1×10¹⁴vp/kg (vector or virus particles per kilogram) systemically administeredcauses no mortality and no visible clinical symptoms in immune deficientor immune competent mice.

SVV elicits efficacy at doses as low as 1×10⁸ vp/kg; therefore, a veryhigh therapeutic index of >100,000 is achieved. Efficacy is very robustin that 100% of large pre-established tumors in mice can be completelyeradicated (see Example 11). This efficacy may be mediated with a singlesystemic injection of SVV without any adjunct therapy. Furthermore, SVVinjected mice show neither clinical symptoms nor recurrence of tumorsfor at least 200 days following injection. SVV can also be purified tohigh titer and can be produced at >200,000 virus particles per cell inpermissive cell lines. SVV-based viral therapy therefore showsconsiderable promise as a safe, effective and new line of treatment forselected types of cancers. Further, SVV has a small and easilymanipulatable genome, simple and fast lifecycle, and a well-understoodbiology of replication, and thus is amenable to modification. Theseproperties, at least in part, allow for methods that generate modifiedSVVs that have new cell or tissue specific tropisms, such that SVV-basedtherapy can be directed to new tumor types resistant to infection by theoriginal SVV isolate.

Accordingly, the present invention provides an isolated nucleic acidcomprising a nucleic acid sequence having at least 65%, 70%, 75%, 80%,85%, 90%, 95% or 99% sequence identity to SEQ ID NOS: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 168, or a contiguous portion of any one of thesesequences that is at least 50 nucleotides in length, or 95% identical toa contiguous portion of any one of these sequences that is at least 10,15 or 20 nucleotides in length. The isolated nucleic acids of theinvention can be RNA or DNA.

For all aspects of the invention, an isolated nucleic acid can comprisea nucleic acid sequence having at least about 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a contiguousportion of any one of the SVV nucleic acid SEQ ID NO sequences herein,wherein the contiguous portion is at least about 20, 25, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 2000 or2500 nucleotides in length, for example. The SVV nucleic acid SEQ ID NOsequences include, for example, SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21 and 168.

For all aspects of the invention, an isolated protein or peptide cancomprise an amino acid sequence having at least about 50%, 55%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity toa contiguous portion of any one of the SVV amino acid SEQ ID NOsequences herein, wherein the contiguous portion is at least at leastabout 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125,150, 175, 200, 225, 250, 275, 300, or 350 amino acids in length, forexample. The SVV amino acid SEQ ID NO sequences include, for example,SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 169.

In another aspect, the invention provides an isolated nucleic acidcomprising a nucleic acid sequence having at least 95%, 96%, 97%, 98%,or 99% sequence identity to SEQ ID NO:168, or to a contiguous portion ofSEQ ID NO:168 that is at least 20, 50, 100, or 200 nucleotides inlength. The isolated nucleic acids can comprise specific portions of SEQID NO:168, including but not limited to: the 5′ untranslated region(UTR) of SVV spanning nucleotides 1-666 of SEQ ID NO:168; the codingsequence for the SVV polyprotein spanning nucleotides 667-7209 of SEQ IDNO:168; the coding sequence for the leader peptide of SVV spanningnucleotides 667-903 of SEQ ID NO:168; the coding sequence for the SVVVP4 protein spanning nucleotides 904-1116 of SEQ ID NO:168; the codingsequence for the SVV VP2 protein spanning nucleotides 1117-1968 of SEQID NO:168; the coding sequence for the SVV VP3 protein spanningnucleotides 1969-2685 of SEQ ID NO:168; the coding sequence for the SVVVP1 protein spanning nucleotides 2686-3477 of SEQ ID NO:168; the codingsequence for the SVV 2A protein spanning nucleotides 3478-3504 of SEQ IDNO:168; the coding sequence for the SVV 2B protein spanning nucleotides3505-3888 of SEQ ID NO:168; the coding sequence for the SVV 2C proteinspanning nucleotides 3889-4854 of SEQ ID NO:168; the coding sequence forthe SVV 3A protein spanning nucleotides 4855-5124 of SEQ ID NO:168; thecoding sequence for the SVV 3B protein spanning nucleotides 5125-5190 ofSEQ ID NO:168; the coding sequence for the SVV 3C protein spanningnucleotides 5191-5823 of SEQ ID NO:168; the coding sequence for the SVV3D protein spanning nucleotides 5824-7209 of SEQ ID NO:168; and the3′UTR of SVV spanning nucleotides 7210-7280 of SEQ ID NO:168.

In one aspect, the invention provides methods for using the SVV 2A, SVVleader peptide, or other SVV proteins or peptide portions thereof, toshut off host cell protein translation. In one aspect, such SVV proteinscan be used to shut off host cell protein translation by interfering orinhibiting with the cap binding protein complex in the host cell.

In another aspect, the invention provides methods for using SVV 2A orother SVV proteins or peptide portions thereof in order to cleave apeptide or protein.

In other aspects, the invention provides an isolated nucleic acid thathybridizes under conditions of high, moderate stringency or lowstringency to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, orto a contiguous portion of any one of these sequences that is at least50 nucleotides in length.

In another aspect, the invention provides a vector comprising a nucleicacid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%sequence identity to SEQ ID NOS 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,168, or to a contiguous portion of any one of these sequences that is atleast 50 nucleotides in length. Vector compositions can also comprisethe nucleic acid regions of SEQ ID NO:168 that code for SVV proteins.

The present invention also provides an isolated polypeptide encoded by anucleic acid having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%sequence identity to a nucleic acid sequence comprising SEQ ID NOS: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or to a contiguous portion of anyone of these sequences that is at least 50 nucleotides in length. Theinvention also provides an isolated polypeptide encoded by a nucleicacid having at least 95%, 96%, 97%, 98%, or 99% sequence identity to anucleic acid region of SEQ ID NO:168 that encodes a SVV protein.

In one aspect, the invention provides an isolated polypeptide comprisingan amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%or 99% sequence identity to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 169, or to a contiguous portion of any one of these sequencesthat is at least 10 amino acids in length.

In another aspect, the invention provides an isolated polypeptidecomprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or99% sequence identity to a contiguous portion of SEQ ID NO:169 that isat least 9, 10, 15, 20 or 50 amino acids in length. Exemplary contiguousportions of SEQ ID NO:169, include but are not limited to, regions thatcomprise a SVV protein, such as: the leader peptide spanning residues1-79; VP4 spanning residues 80-150; VP2 spanning residues 151-434; VP3spanning residues 435-673; VP1 spanning residues 674-937; 2A spanningresidues 938-946; 2B spanning residues 947-1074; 2C spanning residues1075-1396; 3A spanning residues 1397-1486; 3B spanning residues1487-1508; 3C spanning residues 1509-1719; and 3D spanning residues1720-2181.

In another aspect, the invention provides an isolated antibody whichspecifically binds a polypeptide comprising an amino acid sequencehaving at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequenceidentity to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 169, orto a contiguous portion of any one of these sequences that is at least9, 10, 15, or amino acids in length. The isolated antibody can begenerated such that it binds to any protein epitope or antigen of SEQ IDNOS:2 or 169. Further, the antibody can be a polyclonal antibody, amonoclonal antibody or a chimeric antibody.

In one aspect, the invention provides an isolated SVV or derivative orrelative thereof, having a genomic sequence comprising a sequence thatis at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ IDNO:1 or SEQ ID NO:168.

In another aspect, the invention provides an isolated virus having allthe identifying characteristics and nucleic acid sequence of AmericanType Culture Collection (ATCC) Patent Deposit number PTA-5343. Some ofthe viruses of the present invention are directed to the PTA-5343isolate, variants, homologues, relatives, derivatives and mutants of thePTA-5343 isolate, and variants, homologues, derivatives and mutants ofother viruses that are modified in respect to sequences of SVV (bothwild-type and mutant) that are determined to be responsible for itsoncolytic properties.

The present invention further provides an isolated SVV comprising thefollowing characteristics: a single stranded RNA genome (positive (+)sense strand) of ˜7.5 or of ˜7.3 kilobases (kb); a diameter of ˜27nanometers (nm); a capsid comprising at least 3 proteins that haveapproximate molecular weights of about 31 kDa, 36 kDa and 27 kDa; abuoyant density of approximately 1.34 g/mL on cesium chloride (CsCl)gradients; and replication competence in tumor cells. In this aspect,the 31 kDa capsid protein (VP1) can comprise an amino acid sequence thatis at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ IDNO:8 or residues 674-937 of SEQ ID NO:169; the 36 kDa capsid protein(VP2) can comprise an amino acid sequence that is at least 65%, 70%,75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:4 or residues151-434 of SEQ ID NO:169; and the 27 kDa capsid protein (VP3) cancomprise an amino acid sequence that is at least 65%, 70%, 75%, 80%,85%, 90%, 95% or 99% identical to SEQ ID NO:6 or residues 435-673 of SEQID NO:169.

In another aspect, the invention provides an isolated SVV derivative orrelative comprising the following characteristics: replicationcompetence in tumor cells, tumor-cell tropism, and lack of cytolysis innormal cells. An SVV relative includes SVV-like picornaviruses,including viruses from the following USDA isolates: MN 88-36695, NC88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. If anSVV-like picornavirus does not naturally have the characteristics ofreplication competence in tumor cells, tumor-cell tropism, and lack ofcytolysis in non-tumor cells, then the SVV-like picornavirus can bemutated such that these characteristics are obtained. Such mutations canbe designed by comparing the sequence of the SVV-like picornavirus toSVV, and making mutations into the SVV-like picornavirus such that itsamino acid sequence is identical or substantially identical (in aparticular region) to SVV. In another aspect, the virus is replicationcompetent in tumor cell types having neuroendocrine properties.

In other aspects, the present invention provides: a pharmaceuticalcomposition comprising an effective amount of a virus of the inventionand a pharmaceutically acceptable carrier; a cell comprising a virus ofthe invention; a viral lysate containing antigens of a virus of theinvention; and an isolated and purified viral antigen obtained from avirus of the invention.

In yet another aspect, the invention provides a method of purifying avirus of the invention, comprising: infecting a cell with the virus;harvesting cell lysate; subjecting cell lysate to at least one round ofgradient centrifugation; and isolating the virus from the gradient.

In another aspect, the invention provides a method for treating cancercomprising administering an effective amount of a virus or derivativethereof, so as to treat the cancer, wherein the virus has a genomicsequence that comprises a sequence that is at least 65%, 70%, 75%, 80%,85%, 90%, 95% or 99% identical to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 168, or to a portion of SEQ ID NO:1 or SEQ ID NO:168. In oneaspect, the invention provides a method for treating cancer a method fortreating cancer comprising administering an effective amount of a virusor derivative thereof, so as to treat the cancer, wherein the virus hasa genomic sequence that is at least 95%, 96%, 97%, 98%, or 99% identicalto SEQ ID NO:1. The virus that has a genomic sequence that is at least95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 can be, for example,a SVV mutant, a SVV-like picornavirus, or a cardiovirus. The SVV-likepicornavirus can be, for example, a virus from one of the followingisolates MN 88-36695, NC 88-23626, IA 89-47752, NJ 90-10324, IL92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN99197; and SC 363649. The SVV-like picornaviruses can be wild-type ormutant.

In another aspect, the invention provides a method for treating cancercomprising administering an effective amount of a virus comprising acapsid encoding region that comprises a sequence that is at least 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNOS:3, 5, 7, nucleotides 904-3477 of SEQ ID NO:169, or to a contiguousportion thereof that is at least 75, 100, 200, or 500 nucleotides inlength. The invention also provides a method for treating cancercomprising administering an effective amount of a virus comprising acapsid that comprises an amino acid sequence that is at least 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4,6, 8, residues 80-937 of SEQ ID NO:169, or a contiguous portion thereofthat is at least 25, 50, or 100 amino acids in length.

In one aspect, the present invention provides a method for inhibitingcancer progression comprising contacting a cancer cell with a virus orderivative thereof, wherein the virus or derivative thereof specificallybinds to the cancerous cell, wherein the virus has a genomic sequencethat comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%,95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, or 168.

In another aspect, the invention provides a method for inhibiting cancerprogression comprising contacting a cancer cell with a virus orderivative thereof, wherein the virus or derivative thereof specificallyinfects the cancerous cell, wherein the virus has a genomic sequencethat comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 168, or to a contiguous portion of SEQ ID NO:168that is at least 50, 100, 200, or 500 nucleotides in length.

In another aspect, the present invention provides a method for killingcancer cells comprising contacting a cancer cell with an effectiveamount of a virus or derivative thereof, wherein the virus has a genomicsequence that comprises a sequence that is at least 65%, 70%, 75%, 80%,85%, 90%, 95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, or 168.

In another aspect, the present invention provides a method for killingcancer cells comprising contacting a cancer cell with an effectiveamount of a virus or derivative thereof,

wherein the virus has a genomic sequence that comprises a sequence thatis at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:168, or toa contiguous portion of SEQ ID NO:168 that is at least 50, 100, 200 or500 nucleotides in length.

In these methods directed to cancer, the virus can be a picornavirus.The picornavirus can be a cardiovirus, erbovirus, aphthovirus,kobuvirus, hepatovirus, parechovirus, teschovirus, enterovirus,rhinovirus, SVV, or an SVV-like picornavirus. The cardiovirus can beselected from the group consisting of: vilyuisk human encephalomyelitisvirus, Theiler's murine encephalomyelitis virus, andencephalomyocarditis virus. The SVV can be a virus having the ATCCdeposit number PTA-5343 or a virus comprising a nucleic acid sequencethat is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO:1 or SEQ ID NO:168, or to a contiguousportion thereof that is at least 50, 100, 200, or 500 nucleotides inlength. The SVV-like picornavirus can be a virus comprising a nucleicacid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO:168, or to a contiguous portion thereof that is at least 50,100, 200, or 500 nucleotides in length. The SVV-like picornavirus canbe, for example, a virus from one of the following isolates MN 88-36695,NC 88-23626, IA 89-47752, NJ 90-10324, IL 92-48963, CA 131395; LA 1278;IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. TheSVV-like picornaviruses can be wild-type or mutant.

The present invention also provides a method of killing an abnormallyproliferative cell comprising contacting the cell with a virus of theinvention. In one aspect, the abnormally proliferative cell is a tumorcell. In various aspects of this method, the tumor cell is selected fromthe group consisting of: human small cell lung cancer, humanretinoblastoma, human neuroblastoma, human medulloblastoma, mouseneuroblastoma, Wilms' tumor, and human non-small cell lung cancer.

The present invention also provides a method of treating a neoplasticcondition in a subject comprising administering to the subject aneffective amount of a virus of the invention to the mammal. In oneaspect, the neoplastic condition is a neuroendocrine cancer. In anotheraspect, the subject is a mammal. In another aspect, the mammal is ahuman.

The present invention also provides a method of producing a virus of theinvention, comprising: culturing cells infected with the virus underconditions that allow for replication of the virus and recovering thevirus from the cells or the supernatant. In one aspect of this method,the cells are PER.C6 cells. In another aspect of this method, the cellsare H446 cells. In the various aspects of this method, the cells mayproduce over 200,000 virus particles per cell.

In another aspect, the present invention provides a method for detectinga virus of the invention, comprising: isolating RNA from test materialsuspected to contain the virus of the invention; labeling RNAcorresponding to at least 15 contiguous nucleotides of SEQ ID NO:1 orSEQ ID NO:168; probing the test material with the labeled RNA; anddetecting the binding of the labeled RNA with the RNA isolated from thetest material, wherein binding indicates the presence of the virus. Inanother aspect, the present invention provides a nucleic acid probecomprising a nucleotide sequence corresponding to at least 15 contiguousnucleotides of SEQ ID NO:1 or SEQ ID NO:168, or its complement.

The present invention also provides a method for making an oncolyticvirus, the method comprising: (a) comparing a SVV genomic sequence witha test virus genomic sequence; (b) identifying at least a first aminoacid difference between a polypeptide encoded by the SVV genomicsequence and a polypeptide encoded by the test virus genomic sequence;(c) mutating the test virus genomic sequence such that the polypeptideencoded by the test virus genomic sequence has at least one less aminoacid difference to the polypeptide encoded by the SVV genomic sequence;(d) transfecting the mutated test virus genomic sequence into a tumorcell; and (e) determining whether the tumor cell is lytically infectedby the mutated test virus genomic sequence. In one aspect, the aminoacid(s) mutated in the test virus are amino acids in a structuralregion, such as in the capsid encoding region. In another aspect, theamino acids mutated in the test virus are amino acids in anon-structural region.

In one aspect of the method for making an oncolytic virus, the SVVgenomic sequence is obtained from the isolated SVV having the ATCCdeposit number PTA-5343 or from a virus comprising a sequence that is atleast 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portionthereof. In one aspect, the SVV genomic sequence is obtained from theisolated SVV having the ATCC deposit number PTA-5343 or from a viruscomprising a sequence that is at least 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 168, or a contiguous portion thereof that is atleast 50, 100, 200, or 500 nucleotides in length. In another aspect ofthis method, the step of mutating the test virus genomic sequencecomprises mutating a cDNA having the test virus genomic sequence. Inanother aspect of this method, the step of transfecting the mutated testvirus genomic sequence comprises transfecting RNA, wherein the RNA isgenerated from the cDNA having the mutated test virus genomic sequence.

In another aspect of the method for making an oncolytic virus, the testvirus is a picornavirus. The test picornavirus can be a teschovirus,enterovirus, rhinovirus, cardiovirus, erbovirus, apthovirus, kobuvirus,hepatovirus, parechovirus or teschovirus. In another aspect, the testvirus is a cardiovirus. In another aspect, the test virus is a SVV-likepicornavirus. The SVV-like picornavirus can be, for example, a virusfrom one of the following isolates: MN 88-36695, NC 88-23626, IA89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL94-9356; MN/GA 99-29256; MN 99197; and SC 363649. In another aspect, theamino acid differences identified in the methods for making an oncolyticvirus are between a SVV capsid protein and a test virus capsid proteinsequence. In another aspect for making an oncolytic virus, the testvirus genomic sequence is selected from the group consisting of:Vilyuisk human encephalomyelitis virus, Theiler's murineencephalomyelitis virus, and encephalomyocarditis virus. In anotheraspect, the test virus genomic sequence is selected from anencephalomyocarditis virus. In yet another aspect, theencephalomyocarditis virus, the SVV-like picornavirus, or any other testvirus can be selected from an isolate having a nucleic acid sequencecomprising at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequenceidentity to SVV of ATCC deposit number PTA-5343 or SEQ ID NO:1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof that isat least 50, 100, 200, or 500 nucleotides in length.

In another aspect of the method for making an oncolytic cardiovirus, theamino acid difference between the test virus and SVV is in a capsidprotein region of SVV, wherein the amino acid difference is alignedwithin SVV SEQ ID NO:4, 6, 8, residues 80-937 of SEQ ID NO:169, residues80-150 of SEQ ID NO:169, residues 151-434 of SEQ ID NO:169, residues435-673 of SEQ ID NO:169, or residues 674-937 of SEQ ID NO:169.

The present invention also provides a method for making a mutant virushaving an altered cell-type tropism, the method comprising: (a) creatinga library of viral mutants comprising a plurality of nucleic acidsequences; (b) transfecting the library of viral mutants into apermissive cell, such that a plurality of mutant viruses is produced;(c) isolating the plurality of mutant viruses; (d) incubating anon-permissive cell with the isolated plurality of mutant viruses; and(e) recovering a mutant virus that was produced in the non-permissivecell, thereby making a mutant virus having an altered tropism. In oneaspect, this method further comprises the steps of: (f) incubating therecovered mutant virus in the non-permissive cell; and (g) recovering amutant virus that that was produced in the non-permissive cell. Inanother aspect, the method further comprises iteratively repeating steps(f) and (g). In another aspect, the library of viral mutants is createdfrom a parental sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 168, or a contiguous portion thereof.

In one aspect of the method for making a mutant virus having an alteredcell-type tropism, the incubating is conducted in a multi-wellhigh-throughput platform wherein the platform comprises a differentnon-permissive cell-type in each well. In this aspect, the method canfurther comprise screening the platform to identify which wells containa mutant virus that kills the cells. In another aspect, the screening isconducted by analyzing light absorbance in each well.

In another aspect of the method for making a mutant virus having analtered cell-type tropism, the non-permissive cell is a tumor cell.

In another aspect of the method for making a mutant virus having analtered cell-type tropism, the step of creating the library of viralmutants comprises: (i) providing a polynucleotide having a sequenceidentical to a portion of a genomic sequence of a virus; (ii) mutatingthe polynucleotide in order to generate a plurality of different mutantpolynucleotide sequences; and (iii) ligating the plurality of mutatedpolynucleotides into a vector having the genomic sequence of the virusexcept for the portion of the genomic sequence of the virus that thepolynucleotide in step (i) contains, thereby creating the library ofviral mutants. In one aspect, the genomic sequence of a virus is from apicornavirus. In another aspect, the genomic sequence of a viruscomprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,168, or a contiguous portion thereof. In another aspect, the genomicsequence of a virus comprises a sequence that is at least 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 168, or a contiguous portion thereofthat is at least 50, 100, 200, or 500 nucleotides in length. In oneaspect, the virus that comprises a sequence that is at least 95%, 96%,97%, 98%, or 99% identical to a contiguous portion of SEQ ID NO:168 thatis at least 50, 100, 200, or 500 nucleotides in length is a SVV-likepicornavirus. In another aspect, in the step of creating the library ofviral mutants, the mutating of step (ii) is conducted by randominsertion of nucleotides into the polynucleotide. In one aspect, therandom insertion of nucleotides is conducted bytrinucleotide-mutagenesis (TRIM). In another aspect, at least a portionof the nucleotides inserted into the polynucleotide encodes an epitopetag. In another aspect, in the step of creating the library of viralmutants, the mutating of step (ii) is conducted in a capsid encodingregion of the polynucleotide.

The present invention also provides a method for making a mutant virushaving an altered cell-type tropism, the method comprising: (a) creatinga library of mutant polynucleotide sequences of a virus, wherein thecreating comprises: providing a polynucleotide encoding a capsid regionof the virus; mutating the polynucleotide in order to generate aplurality of different mutant capsid-encoding polynucleotide sequences;and ligating the plurality of mutated capsid-encoding polynucleotidesinto a vector having the genomic sequence of the virus except for thecapsid-encoding region, thereby creating the library of mutantpolynucleotide sequences of the virus; (b) transfecting the library ofmutant polynucleotide sequences into a permissive cell, such that aplurality of mutant viruses is produced; (c) isolating the plurality ofmutant viruses; (d) incubating a non-permissive cell with the isolatedplurality of mutant viruses; and (e) recovering a mutant virus that thatwas produced in the non-permissive cell, thereby making a mutant virushaving an altered tropism. In one aspect, the method further comprisesthe steps of: (f) incubating the recovered mutant virus in thenon-permissive cell; and (g) recovering a mutant virus that that wasproduced in the non-permissive cell. In another aspect, the methodfurther comprises iteratively repeating steps (f) and (g). In anotheraspect, the mutating is conducted by random insertion of nucleotidesinto the capsid-encoding polynucleotide. In another aspect, at least aportion of the nucleotides randomly inserted into the capsid-encodingpolynucleotide encodes an epitope tag. In another aspect, the randominsertion of nucleotides is conducted by TRIM. In another aspect, theplurality of different mutant capsid-encoding polynucleotide sequencescomprises greater than 10⁸ or 10⁹ different capsid-encodingpolynucleotide sequences. The library of mutant polynucleotide sequencescan be from, for example, a cardiovirus or an SVV-like picornavirus.

In one aspect, a method for making a mutant SVV having an alteredcell-type tropism comprises: (a) creating a cDNA library of SVV mutants;(b) generating SVV RNA from the cDNA library of SVV mutants; (c)transfecting the SVV RNA into a permissive cell, such that a pluralityof mutant SVV is produced; (d) isolating the plurality of mutant SVV;(e) incubating a non-permissive tumor cell with the isolated pluralityof mutant SVV; and (f) recovering a mutant SVV that lytically infectsthe non-permissive tumor cell, thereby making a mutant SVV having analtered tropism. In another aspect, the method further comprises thesteps of: (g) incubating the recovered mutant SVV in the non-permissivecell; and (h) recovering a mutant SVV that lytically infects thenon-permissive tumor cell. In another aspect, the method furthercomprises iteratively repeating steps (g) and (h). In one aspect, theincubating is conducted in a multi-well high-throughput platform whereinthe platform comprises a different non-permissive tumor cell-type ineach well. In another aspect, the method further comprises screening theplatform to identify which wells contain a mutant SVV that lyticallyinfects the cells. In another aspect, the screening is conducted byanalyzing light absorbance in each well. In one aspect, the cDNA libraryof SVV mutants comprises a plurality of mutant SVV capsid polynucleotidesequences. In another aspect, the plurality of mutant SVV capsidpolynucleotide sequences is generated by random insertion ofnucleotides. In another aspect, at least a portion of the sequence ofthe nucleotides randomly inserted encodes an epitope tag. In anotheraspect, the random insertion of nucleotides is conducted by TRIM. Inanother aspect, the cDNA library of SVV mutants is generated from a SVVof ATCC deposit number PTA-5343. In another aspect, the cDNA library ofSVV mutants is generated from a SVV comprising a sequence having atleast 99%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% sequence identity to SEQID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a contiguousportion thereof that is at least 50, 100, 200, or 500 nucleotides inlength. In one aspect, the cDNA library of SVV mutants is generated froman SVV comprising a sequence having at least 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:168, or to a contiguous portion thereofthat is at least 50, 100, 200, or 500 nucleotides in length. In anotheraspect, the non-permissive tumor cell is a tumor cell-line or a tumorcell-type isolated from a patient.

The present invention also provides a method for making a mutant virushaving a tumor cell-type tropism in vivo, the method comprising: (a)creating a library of viral mutants comprising a plurality of nucleicacid sequences; (b) transfecting the library of viral mutants into apermissive cell, such that a plurality of mutant viruses is produced;(c) isolating the plurality of mutant viruses; (d) administering theisolated plurality of mutant viruses to a mammal with a tumor, whereinthe mammal is not a natural host of the unmutated form of the mutantvirus; and (e) recovering a virus that replicated in the tumor, therebymaking a mutant virus having a tumor cell-type tropism in vivo. In oneaspect, the step of creating a library of viral mutants comprises:providing a polynucleotide encoding a capsid region of a virus; mutatingthe polynucleotide in order to generate a plurality of different mutantcapsid-encoding polynucleotide sequences; and ligating the plurality ofmutated capsid-encoding polynucleotides into a vector having the genomicsequence of the virus except for the capsid-encoding region, therebycreating the library of viral mutants. In another aspect, the virusrecovered in step (e) lytically infects cells of the tumor. In anotheraspect for a method for making a mutant virus having a tumor cell-typetropism in vivo, the tumor is a xenograft, a syngeneic tumor, anorthotopic tumor or a transgenic tumor. In another aspect, the mammal isa mouse.

For all the methods of the present invention, the virus can be apicornavirus. The picornavirus can be a cardiovirus, erbovirus,aphthovirus, kobuvirus, hepatovirus, parechovirus, teschovirus,entrovirus, rhinovirus, or a virus belonging to the genus to which SVVbelongs. The virus can be a cardiovirus. The virus can be an SVV-likepicornavirus. The virus can be SVV. The SVV can be a SVV having the ATCCPatent Deposit No. PTA-5343 or a SVV comprising a sequence that is atleast 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:1,3, 6, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portionthereof. Further, the cardiovirus can be selected from the groupconsisting of: vilyuisk human encephalomyelitis virus, Theiler's murineencephalomyelitis virus, and encephalomyocarditis virus. In one aspect,the SVV-like picornavirus is selected from the group of isolatesconsisting of: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN99197; and SC 363649. In another aspect, the present inventionencompasses any virus that is selected from an isolate having at least99%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% sequence identity to SVV ofATCC deposit number PTA-5343 or SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 168, or a contiguous portion thereof or is otherwise consideredrelated to SVV to by sequence homology.

In another aspect, the present invention encompasses any virus having atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleicacid sequence identity to SVV of ATCC deposit number PTA-5343, to SEQ IDNO:168, or to a contiguous portion of SEQ ID NOS: 1 or 168 that is atleast 100, 200, 300, 400, 500, 750, 1000, 1500, or 2000 nucleotides inlength.

The present invention also provides an oncolytic virus made by any ofthe methods for making a mutant virus disclosed herein. In one aspect,the present invention provides a method for treating a patient with anoncolytic virus, the method comprising: (a) inactivating an oncolyticvirus made by any of the methods for making a mutant virus disclosedherein, such that the oncolytic virus is non-infectious and the tropismof the oncolytic virus is unaffected; and (b) administering theirradiated oncolytic virus to a patient afflicted with a tumor. Inanother aspect, the method for treating a patient further comprisesattaching a toxin to the inactivated oncolytic virus.

In another aspect, the present invention provides a method for treatinga patient with a tumor with SVV, the method comprising: (a) inactivatinga SVV such that the virus is non-infectious and the tropism isunaffected; and (b) administering the inactivated SVV in a patientafflicted with a tumor. In another aspect, the method for treating apatient with a tumor with SVV further comprises attaching a toxin to theinactivated SVV.

In another aspect, the present invention provides a SVV compositioncomprising an inactivated SVV or attenuated SVV. In another aspect, thepresent invention provides a SVV comprising an epitope tag incorporatedin the capsid region.

The present invention also provides a method for treating a patient witha tumor with SVV, the method comprising: (a) creating a mutant SVVcomprising an epitope tag encoded in the capsid; (b) attaching a toxinto the epitope tag; and (c) administering the mutant SVV with theattached toxin to a patient afflicted with a tumor. In one aspect, thecreating comprises: inserting an oligonucleotide encoding an epitope taginto a capsid-encoding region polynucleotide of SVV. In one aspect, themutant SVV does not have an altered cell-type tropism. In anotheraspect, the method further comprises inactivating the mutant SVV suchthat the mutant SVV is not infectious or cannot replicate.

The present invention also provides a method for detecting a tumor cellin a sample comprising: (a) isolating a tumor sample from a patient; (b)incubating the tumor sample with an epitope-tagged SVV; and (c)screening the tumor sample for bound SVV by detecting the epitope tag.

In one aspect, the invention provides a method for detecting a tumorcell in vivo comprising: (a) administering to a patient an inactivatedepitope-tagged SVV, wherein a label is conjugated to the epitope-tag;and (b) detecting the label in the patient. In the methods for detectinga tumor cell of the present invention, the SVV can be a mutant SVVgenerated by the methods disclosed herein.

In one aspect, the invention provides a method for detecting a tumorcell in a sample comprising: (a) isolating a cell sample from a subject;(b) incubating the cell sample with SVV (or an SVV-like picornavirus);(c) incubating the cell sample from step (b) with an antibody specificto SVV (or an antibody specific to an SVV-like picornavirus); and (d)screening the cell sample for bound antibody, wherein bound antibodyindicates that the sample contains a tumor cell.

In one aspect, the invention provides a method for determining whether asubject is candidate for SVV therapy, the method comprising: (a)isolating a cell from the subject; (b) incubating the cell with SVV; (c)incubating the sample from step (b) with an anti-SVV antibody; and (d)detecting for the presence of the anti-SVV antibody on or in the cell,wherein a positive detection indicates that the subject is a candidatefor SVV therapy.

Screening a cell sample for bound antibody or detecting for the presenceof an anti-SVV antibody can be conducted by adding a secondary antibodythat can bind to the constant regions or non-epitope binding regions ofthe anti-SVV antibody, wherein the secondary antibody is conjugated orlabeled with a detectable marker. The detectable marker can be, forexample, a fluorophore such as fluorescein. When a secondary antibody islabeled with a detectable marker, the detectable marker can be detected,for example, by fluorescent microscopy. The cell from the subject can befrom a tissue biopsy from the subject. The tissue biopsy can be from atumor in the subject or from a region in the subject that is suspectedto contain tumor cells. SVV directly labeled with fluorophore can alsobe used in identification of tumor cells.

Further, the methods for treating neoplastic conditions, for detectingneoplastic conditions and for producing SVV, apply to wild-type SVV,mutant (including modified or variant) SVV, relatives of SVV, SVV-likepicornaviruses, and other tumor-specific viruses of the invention.

The viruses of the present invention, and the compositions thereof, canbe used in the manufacture of a medicament for treating the diseasesmentioned herein. Further, the viruses and composition thereof of theinvention can be used for the treatment of the diseases mentionedherein. Thus, in one aspect of the present invention, the presentinvention provides the use of SVV (or mutants, derivatives, relatives,and compositions thereof) for the treatment of cancer or in themanufacture of a medicament for treating cancer.

SVV and SVV-like viruses for gene therapy: Replication defective SVVexpressing gene(s) of interest can be used to deliver genes to correctgenetic disorders. SVV and SVV-like viruses can also be used as deliveryvehicle for siRNA to prevent any specific gene expression. Replicationdefective viruses can be grown in complementing cell lines and/or in thepresence of a helper virus to provide for missing functions in therecombinant virus.

IRES of picornaviruses known to play a role in expression of genes in atissue specific manner. IRES of SVV and SVV-like viruses can be used toreplace IRES of other picornaviruses. This strategy can be used togenerate viruses with altered tissue tropism. In one aspect, theinvention provides an IRES of SVV or an IRES from an SVV-related virusfor the purpose of expressing two genes from a single promoter in atissue specific manner.

Self-cleavage properties of 2A protease of SVV can be used to expressmore than one gene in equal amounts using single promoter andtranscription termination signal sequences. In one aspect, the inventionprovides a self-cleaving 2A peptide of SVV or of an SVV-related virusfor the purpose of expressing of two or more proteins in equal amountsunder the control of single promoter and a single poly(A) signal. Inanother aspect, the invention provides the use of an SVV or anSVV-related virus 3C protease to cleave polypeptides for production ofproteins from a eukaryotic cell. In another aspect, the inventionprovides for the use of an SVV or an SVV-like virus leader peptide tocause shut off of cell protein synthesis in tumor cells or another celltype of interest.

Virus like particles of SVV can be generated and used as vaccines andidentify a particular cell type in a mixed population of cells.

Deposit Information

The following material has been deposited with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va., 20110-2209,U.S.A., under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. All restrictions on the availability of the depositedmaterial will be irrevocably removed upon the granting of a patent.Material: Seneca Valley Virus (SVV). ATCC Patent Deposit Number:PTA-5343. Date of Deposit: Jul. 25, 2003.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of virotherapy using oncolytic viruses.Oncolytic viruses have the properties to replicate, spread and killtumor cells selectively through a tumor mass by locally injecting thevirus or by systemically delivering the virus.

FIG. 2 shows purified SVV stained with uranyl acetate and examined bytransmission electron microscopy. Spherical virus particles are about 27nm in diameter.

FIG. 3 is an electron micrograph of an SVV-infected PER.C6 cell that hasa large crystalline inclusion and large vesicular bodies.

FIG. 4A shows an analysis of SVV RNA. SVV genomic RNA is extracted usingguanidium thiocyanate and a phenol extraction method using Trizol(Invitrogen Corp., Carlsbad, Calif.). RNA is resolved through a 1.25%denaturing agarose gel. The band is visualized by ethidium bromide(EtBr) staining and photographed. In lane 2, a predominant band of SVVgenomic RNA is observed, indicating that the size of the full-length SVVgenome is about 7.5 kilobases.

FIG. 4B is a schematic showing the genome structure and protein productsgenerated from polyprotein processing for picornaviruses, including SVV.

FIGS. 5A-5E presents the nucleotide sequence of SVV (SEQ ID NO:1) andthe encoded amino acid sequence (SEQ ID NO:2). The stop codon isdepicted by a “*” at positions 5671-3. As a general note, in sequencedisclosures that include positions where the exact nucleotide is beingconfirmed, these positions are represented by an “n”. Therefore, incodons that possess an “n”, the relevant amino acid is depicted by a“x”.

FIGS. 6A-6D presents the nucleotide sequence (SEQ ID NO:1) of themajority of the full-length genome of SVV. The nucleotide sequence wasderived from the SVV isolate having the ATCC Patent Deposit Number:PTA-5343. Date of Deposit: Jul. 25, 2003.

FIGS. 7A-7B presents the amino acid sequence (SEQ ID NO:2) encoded bySEQ ID NO:1.

FIG. 8 presents the nucleotide sequence (SEQ ID NO:3) of the partial 1Bor VP2 encoding region of SVV. This sequence is identical to nucleotides4-429 of SEQ ID NO:1.

FIG. 9 presents the amino acid sequence (SEQ ID NO:4) of the partial SVVVP2 protein that is encoded by SEQ ID NO:3. The sequence listed in SEQID NO:4 is identical to amino acids 2-143 of SEQ ID NO:2.

FIG. 10 presents the nucleotide sequence (SEQ ID NO:5) of the 1C or VP3encoding region of SVV. This sequence is identical to nucleotides430-1146 of SEQ ID NO:1.

FIG. 11 presents the amino acid sequence (SEQ ID NO:6) of the SVV VP3protein that is encoded by SEQ ID NO:5. The sequence listed in SEQ IDNO:6 is identical to amino acids 144-382 of SEQ ID NO:2.

FIG. 12 presents the nucleotide sequence (SEQ ID NO:7) of the 1D or VP1encoding region of SVV. This sequence is identical to nucleotides1147-1923 of SEQ ID NO:1.

FIG. 13 presents the amino acid sequence (SEQ ID NO:8) of the SVV VP1protein that is encoded by SEQ ID NO:7. The sequence listed in SEQ IDNO:8 is identical to amino acids 383-641 of SEQ ID NO:2.

FIG. 14 presents the nucleotide sequence (SEQ ID NO:9) of the 2Aencoding region of SVV. This sequence is identical to nucleotides1924-1965 of SEQ ID NO:1.

FIG. 15 presents the amino acid sequence (SEQ ID NO:10) of the SVV 2Aprotein that is encoded by SEQ ID NO:9. The sequence listed in SEQ IDNO:10 is identical to amino acids 642-655 of SEQ ID NO:2.

FIG. 16 presents the nucleotide sequence (SEQ ID NO:11) of the 2Bencoding region of SVV. This sequence is identical to nucleotides1966-2349 of SEQ ID NO:1.

FIG. 17 presents the amino acid sequence (SEQ ID NO:12) of the SVV 2Bprotein that is encoded by SEQ ID NO:11. The sequence listed in SEQ IDNO:12 is identical to amino acids 656-783 of SEQ ID NO:2.

FIG. 18 presents the nucleotide sequence (SEQ ID NO:13) of the 2Cencoding region of SVV. This sequence is identical to nucleotides2350-3315 of SEQ ID NO:1.

FIG. 19 presents the amino acid sequence (SEQ ID NO:14) of the SVV 2Cprotein that is encoded by SEQ ID NO:13. The sequence listed in SEQ IDNO:14 is identical to amino acids 784-1105 of SEQ ID NO:2.

FIG. 20 presents the nucleotide sequence (SEQ ID NO:15) of the 3Aencoding region of SVV. This sequence is identical to nucleotides3316-3585 of SEQ ID NO:1.

FIG. 21 presents the amino acid sequence (SEQ ID NO:16) of the SVV 3Aprotein that is encoded by SEQ ID NO:15. The sequence listed in SEQ IDNO:16 is identical to amino acids 1106-1195 of SEQ ID NO:2.

FIG. 22 presents the nucleotide sequence (SEQ ID NO:17) of the 3Bencoding region of SVV. This sequence is identical to nucleotides3586-3651 of SEQ ID NO:1.

FIG. 23 presents the amino acid sequence (SEQ ID NO:18) of the SVV 3Bprotein that is encoded by SEQ ID NO:17. The sequence listed in SEQ IDNO:18 is identical to amino acids 1196-1217 of SEQ ID NO:2.

FIG. 24 presents the nucleotide sequence (SEQ ID NO:19) of the 3Cencoding region of SVV. This sequence is identical to nucleotides3652-4284 of SEQ ID NO:1.

FIG. 25 presents the amino acid sequence (SEQ ID NO:20) of the SVV 3Cprotein that is encoded by SEQ ID NO:19. The sequence listed in SEQ IDNO:20 is identical to amino acids 1218-1428 of SEQ ID NO:2.

FIG. 26 presents the nucleotide sequence (SEQ ID NO:21) of the 3Dencoding region of SVV. This sequence is identical to nucleotides4285-5673 of SEQ ID NO:1.

FIG. 27 presents the amino acid sequence (SEQ ID NO:22) of the SVV 3Dprotein that is encoded by SEQ ID NO:21. The sequence listed in SEQ IDNO:22 is identical to amino acids 1429-1890 of SEQ ID NO:2.

FIGS. 28A-28H present an amino acid sequence alignment between SVV SEQID NO:2 and various members of the Cardiovirus genus, such asEncephalomyocarditis virus (EMCV; species Encephalomyocarditis virus),Theiler's murine encephalomyocarditis virus (TMEV; species Theilovirus),a rat TMEV-like agent (TLV; species Theilovirus), and Vilyuisk humanencephalomyelitis virus (VHEV; species Theilovirus). The specificsequences of the various Cardioviruses are presented in: SEQ ID NOS: 23(EMCV-R), 24 (EMCV-PV21), 25 (EMCV-B), 26 (EMCV-Da), 27 (EMCV-Db), 28(EMCV-PV2), 29 (EMCV-Mengo), 30 (TMEV/DA), 31 (TMEV/GDVII), 32(TMEV/BeAn8386), 33 (TLV-NGS910) and 34 (VHEV/Siberia-55).

Number positions in FIG. 28 do not correspond to the numbering of thesequence listings. The “I” symbol indicates cleavage sites where thepolyprotein is cleaved into its final functional products. For example,the alignment between positions 1 and 157 is in the 1A (VP4) region. Thealignment between positions: 159 and 428 is in the 1B (VP2) region; 430and 668 is in the 1C (VP3) region; 670 and 967 is in the 1D (VP1)region; 969 and 1111 is in the 2A region; 1112 and 1276 is in the 2Bregion; 1278 and 1609 is in the 2C region; 1611 and 1700 is in the 3Aregion; 1702 and 1723 is in the 3B region; 1725 and 1946 is in the 3Cregion; 1948 and 2410 is in the 3D region. The alignment also showsregions of potential conservation or similarity between the viralsequences as can be determined by standard sequence analysis programs.The alignments were generated using BioEdit 5.0.9 and Clustal W 1.81.

FIG. 29 lists the final polypeptide products of SVV with respect to SEQID NO:2. Some conserved motifs are bolded and underlined: 2A/2B“cleavage” (NPGP (SEQ ID NO:111)); 2C ATP-binding (GxxGxGKS/T (SEQ IDNO:112) and hyhyhyxxD); 3B (VPg)/RNA attachment residue (Y); 3C (pro)active site residues (H, C, H); 3D (pol) motifs (KDEL/IR (SEQ IDNO:113), PSG, YGDD (SEQ ID NO:114), FLKR (SEQ ID NO:115)).

FIG. 30 lists the picornavirus species that were used in sequenceanalyses with SEQ ID NOS:1 and 2 to determine the phylogeneticrelationship between SVV and these picornaviruses (see Example 4, PartI).

FIG. 31 shows the phylogenetic relationship between SVV (SEQ ID NO:4)and other picornaviruses in view of VP2 sequence analyses. The figureshows a bootstrapped neighbor-joining tree (see Example 4, Part I).

FIG. 32 shows a bootstrapped neighbor-joining tree for VP3 between SVV(SEQ ID NO:6) and other picornaviruses (see Example 4, Part I).

FIG. 33 shows a bootstrapped neighbor-joining tree for VP1 between SVV(SEQ ID NO:8) and other picornaviruses (see Example 4, Part I).

FIG. 34 shows a bootstrapped neighbor-joining tree for P1 (i.e., 1A, 1B,1C and 1D) between SVV (i.e., partial P1—amino acids 2-641 of SEQ IDNO:2) and other picornaviruses (see Example 4, Part I).

FIG. 35 shows a bootstrapped neighbor-joining tree for 2C between SVV(SEQ ID NO:14) and other picornaviruses (see Example 4, Part I).

FIG. 36 shows a bootstrapped neighbor-joining tree for 3C (pro) betweenSVV (SEQ ID NO:20) and other picornaviruses (see Example 4, Part I).

FIG. 37 shows a bootstrapped neighbor-joining tree for 3D (pol) betweenSVV (SEQ ID NO:22) and other picornaviruses (see Example 4, Part I).

FIG. 38 presents the actual amino acid percent identities of VP2 betweenSVV (SEQ ID NO:4) and other picornaviruses (see Example 4, Part I).

FIG. 39 presents the actual amino acid percent identities of VP3 betweenSVV (SEQ ID NO:6) and other picornaviruses (see Example 4, Part I).

FIG. 40 presents the actual amino acid percent identities of VP1 betweenSVV (SEQ ID NO:8) and other picornaviruses (see Example 4, Part I).

FIG. 41 presents the actual amino acid percent identities of P1 betweenSVV (partial capsid or P1—amino acids 2-641 of SEQ ID NO:2) and otherpicornaviruses (see Example 4, Part I).

FIG. 42 presents the actual amino acid percent identities of 2C betweenSVV (SEQ ID NO:14) and other picornaviruses (see Example 4, Part I).

FIG. 43 presents the actual amino acid percent identities of 3C (pro)between SVV (SEQ ID NO:20) and other picornaviruses (see Example 4, PartI).

FIG. 44 presents the actual amino acid percent identities of 3D (pol)between SVV (SEQ ID NO:22) and other picornaviruses (see Example 4, PartI).

FIG. 45 shows the VP2 (˜36 kDa), VP1 (˜31 kDa) and VP3 (˜27 kDa)proteins of SVV as analyzed by SDS-PAGE. Purified SVV was subjected toSDS-PAGE and proteins were visualized by silver stain. Lane “MWt” ismolecular weight markers; lane “SVV” contains structural proteins ofSVV. The sizes of three molecular weight markers and the names of viralproteins are also given.

FIGS. 46A-46B show the amounts of SVV in blood and tumor followingsystemic administration (Example 7). H446 tumor bearing nude mice weretreated with SVV at a dose of 1×10¹²/kg by tail vein injection. The micewere bled at 0, 1, 3, 6, 24, 48, 72 hours and at 7 days post-injection,and the plasma was separated from the blood immediately aftercollection, diluted in infection medium, and used to infect PER.C6cells. The tumors were harvested at 6, 24, 48, 72 hours and at 7 dayspost-injection. The tumors were cut into small sections and suspended inone mL of medium and CVL was made.

FIGS. 46C-46D presents data relating to SVV clearance in vivo. Thefigures show that SVV exhibits a substantially longer resident time inthe blood compared to similar doses of i.v. adenovirus (Example 7), andtherefore SVV has a slower clearance rate than adenovirus in vivo.Following a single intravenous (i.v.) dose, SVV remains present in theblood for up to 6 hours (FIG. 46C; FIG. 46C is a duplication of FIG. 46Afor comparison purposes to FIG. 46D), whereas adenovirus is cleared ordepleted from the blood in about an hour (FIG. 46D).

FIG. 47 shows immunohistochemistry and hematoxylin and eosin (H&E)staining of H446 xenograft sections (Example 7). H446 tumor bearing nudemice were treated with Hank's balanced salt solution (HBSS) or SVV at adose of 1×10¹² vp/kg by tail vein injection. The mice were sacrificed at3 days post-injection and the tumors were collected. The virus proteinsin the tumor cells are visualized by immunohistochemistry usingSVV-specific mouse antibodies (upper panels). The general morphology ofH446 tumor cells collected from HBSS or SVV treated mice were stained byH&E stain (lower panels).

FIG. 48 shows SVV mediated cytotoxicity in primary human hepatocytes(Example 9). Primary human hepatocytes plated in collagen coated 12-wellplates were infected with SVV at 1, 10 and 100 and 1000 particles percell (ppc). Three days after infection, the cell associated lactatedehydrogenase (LDH) and LDH in the culture supernatant were measuredseparately. Percent cytotoxicity was determined as a ration of LDH unitsin supernatant over maximal cellular LDH plus supernatant LDH.

FIG. 49 shows virus production by SVV in selected cell lines. To assessthe replicative abilities of SVV, selected normal cells and tumor cellswere infected with SVV at one virus particle per cell (ppc) (Example 9).After 72 hours, cells were harvested and CVL was assayed for titer onPER.C6 cells. For each cell line, the efficiency of SVV replication wasexpressed as plaque forming units per milliliter (pfu/ml).

FIG. 50 shows toxicity in nude and CD1 mice according to body weights(Example 10). The mean body weight of mice in each treatment group weremeasured different days post virus administration. Mice were injectedwith a single dose of SVV or PBS by tail vein on day 1.

FIG. 51 shows efficacy in a H446 xenograft model. H446 tumors areestablished in nude mice and the mice are sorted into groups (n=10) andtreated via tail vein injection with either HBSS or six different dosesof SVV (Example 11). On study day 20, five mice from the HBSS group thatbear large tumors (mean tumor volume=2000 mm³) were injected with 1×10¹¹vp/kg (indicated by an arrow). Data is expressed as mean tumorvolume+standard deviation (SD).

FIG. 52 shows a picture of H446 xenograft nude mice that have beenuntreated or treated with SVV (Example 11). The efficacy of SVV is veryrobust in that 100% of large pre-established tumors were completelyeradicated. SVV-treated mice show neither clinical symptoms norrecurrence of tumors for at least 200 days following injection.

FIG. 53 presents data relating to SVV tumor specificity and efficacy invitro (Example 11). The graphs show cell survival following incubationof either H446 human small cell lung carcinoma (SCLC) tumor cells (topgraph) or normal human H460 cells (bottom graph). SVV specificallykilled the tumor cells with an EC₅₀ of approximately 10⁻³ particles percell. In contrast, normal human cells were not killed at anyconcentration of SVV.

FIG. 54 depicts a representative plasmid containing the complete genomeof SVV (Example 15). The presence of the T7 promoter on the vectorupstream of the SVV sequence allows for the in vitro transcription ofthe SVV sequence such that SVV RNA molecules can be generated.

FIG. 55 depicts a schematic for the construction of a full-length andfunctional genomic SVV plasmid and subsequent SVV virus production(Example 16). To obtain a functional genomic SVV clone, the completegenome of a SVV can be cloned into a vector with a T7 promoter. This canbe accomplished by making cDNA clones of the virus, sequencing them andcloning contiguous pieces into one plasmid, resulting in the plasmiddepicted “pSVV”. The plasmid with the full genome of SVV can then bereverse-transcribed to generate SVV RNA. The SVV RNA is then transfectedinto permissive mammalian cells and SVV virus particles can then berecovered and purified.

FIG. 56 depicts a schematic for the construction of a vector (“pSVVcapsid”) containing the coding sequence (i.e., coding regions for 1A-1D)for the SVV capsid (Example 16). The pSVV capsid can then be used togenerate a library of SVV capsid mutants.

FIG. 57 shows one method of mutating the SVV capsid for the generationof a library of SVV capsid mutants (Example 16). The figure illustratesthe insertion of an oligonucleotide sequence at random sites of theplasmid. The oligonucleotides can be random oligonucleotides,oligonucleotides with known sequences, or an oligonucleotide encoding anepitope tag. In the figure, the restriction enzyme CviJI randomlycleaves the pSVV capsid DNA. Linearized pSVV capsid DNA that has beencut at a single site is isolated and purified from a gel, and ligatedwith oligonucleotides.

FIG. 58 presents a scheme to generate a library of full-length SVVmutants comprising sequence mutations in the capsid encoding region(Example 16). For example, the capsid encoding region from a pSVV capsidmutant library (generated according to the scheme depicted in FIG. 57,for example) is isolated by restriction digestion and gel purification.The vector containing the full-length SVV sequence is also digested suchthat the capsid encoding region is cut out. The capsid encoding regionfrom the pSVV capsid mutant library is then ligated to the pSVV vectorthat is missing its wild-type capsid sequence, thereby generating alibrary of full-length SVV mutants (the “pSVVFL” vector) having aplurality of mutations in the capsid encoding region.

FIG. 59 presents a general method for producing the SVV virus particlescomprising a library of capsid mutations (Example 16). The pSVVFL vectoris reverse-transcribed to generate SVV RNA. The SVV RNA is transfectedinto permissive cells, wherein SVV mutant virus particles are produced.The virus particles lyse the cells and a population of SVV virusparticles comprising a plurality of capsid variants, “SVV capsidlibrary,” are isolated.

FIG. 60 shows a general method for screening SVV capsid mutants that canspecifically infect tumor cells while being unable to infect non-tumorcells. The SVV capsid library is incubated with a tumor cell line ortissue of interest. After an initial incubation period, the cells arewashed such that SVV virus particles that were unable to gain entry intothe cells are eliminated. The cells are then maintained in culture untilviral lysis is observed. Culture supernatant is then collected toisolate SVV capsid mutants that were able to lytically infect the tumorcell. These viruses can then be grown-up by infecting a known permissivecell-line prior to a counter-screen. A counter-screen is performed byincubating the SVV capsid mutant viruses that were able to infect thetumor cell with normal cells. Only those viruses that remain unbound inthe supernatant are collected, thereby isolating mutant SVV viruses thathave tumor-specificity. This process can be repeated to further refinethe isolation of SVV tumor-specific viruses.

FIG. 61 shows a traditional method for testing whether virus mutants canbind and/or infect cell lines. Traditional methods require what areoften inefficient methods for growing cell-lines, i.e. flasks, such thata mass-screen of a library of virus mutants in relation to a number ofdifferent cell-lines becomes burdensome.

FIG. 62 shows a high-throughput method of the invention for screeningvirus mutants that have the ability to specifically infect differentcell-lines (Example 16). In this figure, a number of different tumorcell-lines are grown in a 384 well-plate. To each well, a sample of avirus is added (for example, a sample of a SVV capsid library). Fromthose wells which show cytopathic effects, the media is collected suchthat any viruses in the media can be amplified by infecting permissivecell lines (for example, for SVV, H446 or PER.C6) in flasks or largetissue culture plates. The viruses are grown such that the RNA can beisolated and the sequence analyzed to determine the encoded peptidesequence inserted by the oligonucleotide-insertion mutagenesis of thecapsid. The peptide itself can then be tested to determine whether itcan bind to a tumor cell-type specifically.

FIG. 63 shows another high-throughput screening schematic (Example 16).Tumor and normal cell lines are grown in multi-well plates. Viruses areadded to each well to test whether the cells are killed byvirus-mediated lysis. Cytopathic effects can be quickly assayed byreading the light-absorbance in each well. Viruses from the wellsshowing cytopathic effects are grown up and tested in further in vitro(re-testing of tumor and normal cell lines) and in vivo models (testingwhether the virus can kill explanted tumors in mice).

FIG. 64 shows that SVV capsid mutants (SEQ ID NOS: 45-48, respectively,in order of appearance) having new tumor-specific tropisms can beanalyzed to generate tumor-selective peptides. Those SVV capsid mutantsthat enable the specific infection of a tumor cell line are sequenced todetermine the peptide encoded by the oligonucleotide insertion. An aminoacid consensus sequence can thereby be determined from the successfulcapsid mutants. Peptides having the consensus sequence can then betested to determine whether they can bind specifically to the tumorcell-type in question. Tumor-selective peptides can then be attached totoxins or drugs in order to serve as tumor-specific targeting vehicles.

FIG. 65 illustrates that an SVV capsid library can be first tested invivo. Mice (including normal, athymic, nude, CD-1 transgenics, etc.) canbe explanted with a specific tumor. These mice are then injected with aSVV derivative library, such as a SVV capsid library. At certain timepoints, tumor cells are recovered from the mice, such that in those micethat display the elimination of a tumor, viruses will be isolated frominitial tumor samples and grown-up in permissive cell lines.

FIG. 66 shows a clinical testing program for the SVV derivatives of thepresent invention.

FIG. 67 illustrates that SVV derivatives (with new tumor tropisms)encoding epitope tags in their capsid can be used for a variety ofpurposes. They can be used as a screening reagent to detect whether aspecific tumor cell is present in tissue samples by assaying for thepresence of the epitope. Alternatively, toxins or other therapeutics canbe attached to the epitope tag, and the virus then administered topatients. Further, wild-type or derivative SVV can be irradiated orinactivated such that the virus particle itself is used as a therapeuticdevice. Either the virus particle induces cellular apoptosis due to thepresence of apoptosis-inducing peptides, or the particle can have atoxin or some other therapeutic attached such that the virus is used aspecific-targeting delivery device.

FIG. 68 shows the basic life-cycle of the picornavirus.

FIG. 69 shows a comparison of the polypeptide lengths of SVV compared toother picornaviruses.

FIG. 70 lists an amino acid comparison of the picornavirus 2A-like NPG/Pproteins (SEQ ID NOS: 49-110, respectively, in order of appearance). Thesequence for SVV is listed at residues 635-656 of SEQ ID NO:2.

FIGS. 71A and 71B list the amino acid sequence (SEQ ID NO:23) forEMCV-R.

FIGS. 72A and 72B list the amino acid sequence (SEQ ID NO:24) forEMCV-PV21 (Accession CAA52361).

FIGS. 73A and 73B list the amino acid sequence (SEQ ID NO:25) for EMCV-B(Accession P17593).

FIGS. 74A and 74B list the amino acid sequence (SEQ ID NO:26) forEMCV-Da (Accession P17594).

FIGS. 75A and 75B list the amino acid sequence (SEQ ID NO:27) forEMCV-Db.

FIGS. 76A and 76B list the amino acid sequence (SEQ ID NO:28) forEMCV-PV2 (Accession CAA60776).

FIGS. 77A and 77B list the amino acid sequence (SEQ ID NO:29) forEMCV-mengo (Accession AAA46547).

FIGS. 78A and 78B list the amino acid sequence (SEQ ID NO:30) forTMEV/DA (Accession AAA47928).

FIGS. 79A and 79B list the amino acid sequence (SEQ ID NO:31) forTMEV/GDVII (Accession AAA47929).

FIGS. 80A and 80B list the amino acid sequence (SEQ ID NO:32) forTMEV/BeAn8386 (Accession AAA47930).

FIGS. 81A and 81B list the amino acid sequence (SEQ ID NO:33) forTLV-NGS910 (Accession BAC58035).

FIG. 82 lists the amino acid sequence (SEQ ID NO:34) for VHEV/Siberia-55(Accession AAA47931).

FIGS. 83A-83H present the full-length genomic sequence of SVV (SEQ IDNO:168) and the encoded polyprotein amino acid sequence (SEQ ID NO:169),where this full-length genomic sequence was obtained from SVV virusesgrown from the SVV isolate having ATCC Patent Deposit Number PTA-5343.Specific features of the SVV genomic sequence, such as the specificcoding regions for proteins cleaved from the polyprotein sequence aredescribed herein.

FIGS. 84A-84D present the full-length genomic sequence of SVV (SEQ IDNO:168). The sequence was obtained from SVV grown from the SVV isolatehaving ATCC Patent Deposit Number PTA-5343.

FIGS. 85A-85B present the amino acid sequence of the full-lengthpolyprotein of SVV (SEQ ID NO:169) encoded by the nucleotides 667-7209of SEQ ID NO:168.

FIG. 86 provides a phylogenetic analysis or epidemiology of SVV withrespect to the full-length genome and polyprotein sequence of SVV fromSEQ ID NOS:168 and 169. SVV is a unique virus, phylogenetically similarto cardioviruses, but in a separate tree. The SVV-like picornavirusesare most likely in the same tree or genus as SVV due to the high levelof sequence identity between SVV and the SVV-like picornaviruses (seeFIGS. 87-89) and due to the ability of antibodies raised againstSVV-like picornaviruses to bind SVV (and vice versa) (see Example 4,Part III, Serum Studies).

FIGS. 87A-87D show a nucleic acid sequence comparison between SVV andsome SVV-like picornaviruses in the areas of the P1 structural regionand 2A. In particular, the comparison is in the VP2(partial)-VP3-VP1-2A(partial) regions. The listed SVV sequence is SEQ ID NO:170; the listedsequence for isolate IA 89-47752 is SEQ ID NO:171; the listed sequencefor isolate CA 131395 is SEQ ID NO:172; the listed sequence for isolateNC 88-23626 is SEQ ID NO:173; the listed sequence for isolate MN88-36695 is SEQ ID NO:174; the listed sequence for isolate NJ 90-10324is SEQ ID NO:175; the listed sequence for isolate IL 92-48963 is SEQ IDNO:176; the listed sequence for isolate LA 1278 (97-1278) is SEQ IDNO:177; and the listed consensus sequence is SEQ ID NO:178.

FIG. 88 shows a nucleic acid sequence comparison between SVV andisolates IA 89-47752 and CA 131395 in the 2C coding region (partial).The listed SVV sequence is SEQ ID NO:179; the listed sequence forisolate IA 89-47752 is SEQ ID NO:180; the listed sequence for isolate CA131395 is SEQ ID NO:181; and the listed consensus sequence is SEQ IDNO:182.

FIGS. 89A-89B show a nucleic acid sequence comparison between SVV andisolates NC 88-23626, MN 88-36695, IA 89-47752, NJ 90-10324, IL92-48963, LA 97-1278, and CA 131395 in the 3D polymerase coding region(partial) and 3′ UTR region. The listed sequences are SVV (SEQ IDNO:183), NC 88-23626 (SEQ ID NO:184), MN 88-36695 (SEQ ID NO:185), IA89-47752 (SEQ ID NO:186), NJ 90-10324 (SEQ ID NO:187), IL 92-48963 (SEQID NO:188), LA 97-1278 (SEQ ID NO:189), CA 131395 (SEQ ID NO:190), andconsensus sequence (SEQ ID NO:191).

FIGS. 90A-90E show that a single dose of SVV is efficacious in reducingthe size and preventing the growth of explanted tumors in mice. FIG. 90Ashows that SVV can reduce the size and prevent the growth of explantedH446 human SCLC tumors (ED₅₀=0.0007). FIG. 90B shows that SVV can reducethe size and prevent the growth of explanted Y79 human retinoblastomatumors (ED₅₀=0.0007). FIG. 90C shows that SVV can reduce the size andprevent the growth of explanted H69AR human SCLC-MDR (multi drugresistant) tumors (ED₅₀=0.05). FIG. 90D shows that SVV can reduce thesize and prevent the growth of explanted H1299 human HSCLC tumors(ED₅₀=4.8). FIG. 90E shows that SVV can reduce the size and prevent thegrowth of explanted N1E-115 murine neuroblastoma tumors in A/J mice(normal immunocompetent mice) (ED₅₀=0.001).

FIG. 91 show a molecular model of the EMCV and TMEV capsid structures incomparison with the sequence of SVV. A molecular model in conjunctionwith the use of algorithms for antigenic prediction allows for peptidesequences to be chosen for polyclonal antibody generation. β-sheets areshown in brown, α-helices are shown in green, and a 12-mer peptidesequence chosen for polyclonal generation is shown in yellow. Theparticular sequence (in the VP2 region) was chosen because it presentsgood surface exposure according to the model.

FIGS. 92A-92D show the specificity of polyclonal antibodies against SVV.FIG. 92A is a negative control, and presents an immunofluorescence imageof cells infected with SVV that are stained with non-specific anti-mousesera and secondary antibody. FIGS. 92B and 92C show immunofluorescenceimages of cells infected with SVV that are stained with mouse anti-SVVsera diluted 1:50 and secondary antibody (anti-mouse Ig conjugated tofluorescein). FIG. 92D shows that polyclonal anti-SVV antibodies can beused in viral binding assays; the image shows an immunofluorescenceimage of SVV concentrated in an outline around a cell because the cellwas put on ice to prevent SVV internalization.

FIG. 93 shows the results of a neutralization assay of GP102 sera on SVV(see Example 18). The neutralization titer (calculated as the highestdilution that neutralizes the virus is 100%) is 1:100.

FIG. 94 shows the results of a neutralization assay of anti-SVV antiseraon MN 88-36695 (see Example 18). The neutralization titer is 1:560.

FIG. 95A and FIG. 95B depict neighbor-joining trees. These trees wereconstructed using PHYLIP (Phylogeny Inference Package Computer Programsfor Inferring Phylogenies) and show the relationship between SVV andseven SVV-like picornaviruses when comparing sequences from regions inP1 and partial 2A (FIG. 95A) and in the 3′ end of the genome (FIG. 95B).

DETAILED DESCRIPTION OF THE INVENTION

The terms “virus,” “viral particle,” “virus particle,” and “virion” areused interchangeably.

The terms “vector particle” and “viral vector particle” areinterchangeable and are to be understood broadly—for example—as meaninginfectious viral particles that are formed when, e.g., a viral vector ofthe invention is transduced or transfected into an appropriate cell orcell line for the generation of infectious particles.

The terms “derivative,” “mutant,” “variant” and “modified” are usedinterchangeably to generally indicate that a derivative, mutant, variantor modified virus can have a nucleic acid or amino acid sequencedifference in respect to a template viral nucleic acid or amino acidsequence. For example, a SVV derivative, mutant, variant or modified SVVmay refer to a SVV that has a nucleic acid or amino acid sequencedifference with respect to the wild-type SVV nucleic acid or amino acidsequence of ATCC Patent Deposit Number PTA-5343.

An “SVV-like picornavirus” as used herein can have at least about 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SVV atthe nucleotide level (see SEQ ID NO:168, FIG. 84, and FIG. 83 for theSVV full-length genomic sequence), where the sequence comparison is notlimited to a whole-genome analysis, but can be focused on a particularregion of the genome, such as the 5′UTR, structural encoding regions,non-structural encoding regions, 3′UTR, and portions thereof. Theparticular length of the genome for sequence comparison that is adequateto determine relatedness/likeness to SVV is known to one skilled in theart, and the adequate length can vary with respect to the percentage ofidentity that is present. The length for sequence comparison can be, forexample, at least 20, 50, 100, 200, 300, 400, 500, 750, 1000, 1500,2000, or 2500 nucleotides. Where the length is shorter, one skilled inthe art understands, for example, that the identity between sequencescan be higher in order to consider the two sequences to be related.However, such guidance is qualified at least with respect toconsiderations of sequence conservation, in that certain regions of thegenome are more conserved than others between related species.Additionally, if an antiserum generated from a virus can neutralize SVVinfection of an SVV permissive cell line, then the virus is consideredto be an SVV-like picornavirus. Additionally, if an antiserum generatedfrom a virus can neutralize SVV infection of an SVV permissive cellline, and that antiserum can also bind to other viruses (for example, ifthe antiserum can be used in indirect immunofluorescence assays todetect virus), then the other viruses that can be bound by the antiserumare considered to be SVV-like picornaviruses. For purposes of theinvention, SVV-like picornaviruses can include cardioviruses. ExemplarySVV permissive cells or cell lines include, but are not limited to, Y79,NCI-H446, N1E-115, NCI-H1770, NCI-H82, PER.C6®, NCI-H69AR, SK-NEP-1,IMR-32, NCI-H187, NCI-H209, HCC33, NCI-H1184, D283 Med, SK-N-AS, BEKPCB3E1, ST, NCI-H1299, DMS 153, NCI-H378, NCI-H295R, BEK, PPASMC,PCASMC, PAoSMC, NCI-H526, OVCAR-3, NCI-H207, ESK-4, SW-13, 293, Hs 578T,HS 1.Tes, and LOX IMVI.

As used herein, the terms “cancer,” “cancer cells,” “neoplastic cells,”“neoplasia,” “tumor,” and “tumor cells,” are used interchangeably, andrefer to cells that exhibit relatively autonomous growth, so that theyexhibit an aberrant growth phenotype characterized by a significant lossof control of cell proliferation. Neoplastic cells can be malignant orbenign. According to the present invention, one type of preferred tumorcells are those with neurotropic properties.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid or protein sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using a sequencecomparison algorithm such as Protein-Protein BLAST (Protein-ProteinBLAST of GenBank databases (Altschul, S.F., Gish, W., Miller, W., Myers,E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J.Mol. Biol. 215:403-410)) or by visual inspection. The BLAST algorithm isdescribed in Altschul et al., J. Mol. Biol., 215:403-410 (1990), andpublicly available BLAST software is provided through the NationalCenter for Biotechnology Information (NCBI)(http://www.ncbi.nlm.nih.gov/).

For example, as used herein, the term “at least 90% identical to” refersto percent identities from 90 to 100 relative to the referencepolypeptides (or polynucleotides). Identity at a level of 90% or more isindicative of the fact that, assuming for exemplification purposes atest and reference polypeptide length of 100 amino acids are compared,no more than 10% (i.e., 10 out of 100) amino acids in the testpolypeptide differs from that of the reference polypeptide. Similarcomparisons can be made between a test and reference polynucleotide.Such differences can be represented as point mutations randomlydistributed over the entire length of an amino acid sequence or they canbe clustered in one or more locations of varying length up to themaximum allowable, e.g., 10 out of 100 amino acid differences (90%identity). Differences are defined as nucleic acid or amino acidsubstitutions, insertions or deletions. At the level of identities aboveabout 85-90%, the result should be independent of the program and gapparameters set; such high levels of identity can be assessed readily,often without relying on software.

The concepts of “high stringency,” “intermediate stringency” or “lowstringency” refer to nucleic acid hybridization conditions. Highstringency conditions refers to conditions that require a greateridentity between a target's nucleic acid sequence and a probe's nucleicacid sequence in order for annealing or hybridization to occur betweenthe target and the probe. Low stringency conditions refer to conditionsthat require a lower identity between a target's nucleic acid sequenceand a probe's nucleic acid sequence in order for annealing orhybridization to occur between the target and the probe. Stringencyconditions can be controlled by the salt concentration of the buffer orby the temperature at which the hybridization is carried out, wherehigher salt concentrations result in less stringent conditions and wherehigher temperatures result in more stringent conditions. Althoughstringency conditions will vary based on the length and nucleic acidcontent of the sequences undergoing hybridization, representativeconditions of high, intermediate and low stringency are described in thefollowing exemplary conditions. A commonly used hybridization buffer isSSC (sodium chloride sodium citrate) with a 20× stock concentrationcorresponding to 0.3 M trisodium citrate and 3 M NaCl. For highstringency conditions, the working concentration of SSC can be0.1×-0.5×(1.5-7.5 mM trisodium citrate, 15-75 mM NaCl) with thehybridization temperature set at 65° C. Intermediate conditionstypically utilize a 0.5×-2×SSC concentration (7.5-30 mM trisodiumcitrate, 75-300 mM NaCl) at a temperature of 55-62° C. Hybridizationsconducted under low stringency conditions can use a 2×-5×SSCconcentration (30-75 mM trisodium citrate, 300-750 mM NaCl) at atemperature of 50-55° C. Note that these conditions are merely exemplaryand are not to be considered limitations.

Seneca Valley Virus (SVV):

SVV is a novel, heretofore undiscovered RNA virus, and with respect topreviously characterized picornaviruses, SVV is most closely related tomembers from the genus Cardiovirus in the family Picornaviridae (seeInternational Application No. PCT/US2004/031594). The results ofsequence analyses between SVV and other cardioviruses are discussed inPCT/US2004/031594, which is hereby incorporated by reference in itsentirety. Since the time of the sequence analysis of SVV described inPCT/US2004/031594, the Picornavirus Study Group has initiated discussionas to whether SVV will be a member of a new genus. FIG. 86 presents agenetic relationship tree between members of the family Picornaviridae.

From initial sequence comparisons to known picornaviruses (seeInternational Application No. PCT/US2004/031504), there were twophylogenetic classification options: (1) to include SVV as a new speciesin the genus Cardiovirus; or (2) assign SVV to a new genus. At that timeand for the International application, SVV was designated to be a novelmember of the genus Cardiovirus. After further analyses however, it hasbeen found that several characteristics of SVV differ with that ofcardioviruses. For example, some cardiovirus genomes contain an extendedinternal poly(C) tract in their 5′ UTRs. SVV does not contain a poly(C)tract. From the additional 5′ sequence information, the InternalRibosome Entry Sequence (IRES) of SVV has been mapped and compared toother picornaviruses, and it has been determined that the SVV IRES isType IV, whereas cardiovirus IRES's are Type II. The cardioviruses havea long (150 amino acid (aa)) 2A protease while SVV has a short (9 aa) 2Aprotease. The size of this protein as well as others (Leader peptide,3A) differs significantly between SVV and cardioviruses. From the studyof other picornaviruses, it is know that these proteins are likelyinvolved in host cell interactions including tropism and virulence.Lastly, it is now thought that the overall sequences differ too much ina number of genome regions and SVV should therefore be considered toform a new genus. Additionally, multiple unique picornaviruses have beendiscovered at the USDA that are more similar to SVV than SVV is to othercardioviruses. Therefore, it has been decided by the Executive Committeeof the International Committee for the Taxonomy of Viruses (ICVT) basedon recommendations made by the Picornavirus Study Group that SVV willmake up a new species of picornavirus, named Seneca Valley virus.However, currently, SVV and these unique USDA picornaviruses (hereinreferred to as being members of the group of SVV-like picornaviruses)are currently unassigned to any genus.

Several of the SVV-like picornaviruses discovered at the USDA are about95-98% identical to SVV at the nucleotide level (for example, see FIGS.87-89). Antisera against one virus (MN 88-36695) neutralizes SVV, andthis virus is reactive to other antisera that can neutralize SVV. TheSVV-like picornaviruses were isolated from pigs, and thus, pigs arelikely a permissive host for SVV and other SVV-like viruses. Examples ofSVV-like picornaviruses isolated from pigs include, but are not limitedto, the following USDA isolates MN 88-36695, NC 88-23626, IA 89-47552,NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356;MN/GA 99-29256; MN 99197; and SC 363649. SVV-like picornaviruses mayalso include cardioviruses closely related to SVV (as determined bysequence analysis or by cross-reactivity to antibodies raised againstSVV antigens). Thus, for purposes of the present invention, SVV can beconsidered: (1) to be closely related to (or to be a member of) thegenus Cardiovirus of the family Picornaviridae, and (2) to be a memberof a new genus of the family Picornaviridae, where members of the newgenus can include SVV and SVV-like picornaviruses not classified to bemembers of other genuses.

SVV, like cardioviruses, can be distinguished from other picornavirusesby special features of their genome organization, common pathologicalproperties, and the dissociability of their virions at pHs between 5 and7 in 0.1M NaCl (Scraba, D. et al., “Cardioviruses (Picornaviridae),” inEncyclopedia of Virology, 2nd Edition, R. G. Webster and A. Granoff,Editors, 1999). The genome of SVV consists of one single-strandedpositive (+) sense strand RNA molecule having a size of 7,310nucleotides including a poly(A) tail of 30 nucleotides in length (seeFIGS. 83A-83H; FIGS. 84A-84D; SEQ ID NO:168). As SVV is a picornavirus,it has a number of features that are conserved in all picornaviruses:(i) genomic RNA is infectious, and thus can be transfected into cells tobypass the virus-receptor binding and entry steps in the viral lifecycle; (ii) a long (about 600-1200 bp) untranslated region (UTR) at the5′ end of the genome (for SVV, nucleotides 1-666 of SEQ ID NO:168), anda shorter 3′ untranslated region (about 50-100 bp; for SVV, nucleotides7210-7280 of SEQ ID NO:168; (iii) the 5′ UTR contains a clover-leafsecondary structure known as the internal ribosome entry site (IRES)(which can be, for example, from about nucleotide 300 to aboutnucleotide 366 of SEQ ID NO:168); cardioviruses have a Type II IRES andSVV has a Type IV IRES; (iv) the rest of the genome encodes a singlepolyprotein (for SVV, nucleotides 667-7209 of SEQ ID NO:168 encode thepolyprotein (SEQ ID NO:169)) and (v) both ends of the genome aremodified, the 5′ end by a covalently attached small, basic protein,“Vpg,” and the 3′ end by polyadenylation (for SVV, nucleotides 7281-7310of SEQ ID NO:168).

The invention provides the isolated SVV virus (ATCC Patent Depositnumber PTA-5343) and the complete genomic content of SVV therefrom. Atfirst, the largest SVV genomic fragment that was sequenced is anisolated SVV nucleic acid, derived from the PTA-5343 isolate, thatcomprises the majority of the SVV genomic sequence, and is listed inFIGS. 5A-5E and FIGS. 6A-6D, and has the designation of SEQ ID NO:1herein. Translation of this nucleotide sequence shows that the majorityof the single polyprotein of SVV is encoded by SEQ ID NO:1. The aminoacid sequence encoded by nucleotides 1 to 5673 of SEQ ID NO:1 is listedin FIGS. 5A-E and FIGS. 7A-7B has the designation of SEQ ID NO:2 herein.The full-length genome or what appears to be the full-length genome hassince been obtained, and is listed in FIGS. 83A-83H and SEQ ID NO:168.Nucleotides 667-7209 encode the full-length polyprotein of SVV, and theamino acid sequence of the polyprotein is listed in FIGS. 83A-83H andSEQ ID NO:169.

The invention provides isolated (or purified) portions of SEQ ID NO:1,including SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, and isolatedportions of SEQ ID NO:168, including the 5′UTR region (1-666), codingregion for the leader peptide (667-903), coding region for the VP4protein (904-1116), coding region for the VP2 protein (1117-1968),coding region for the VP3 protein (1969-2685), coding region for the VP1protein (2686-3474), coding region for the coding region for the 2Aprotein (3478-3504), coding region for the 2B protein (3505-3888),coding region for the 2C protein (3889-4854), coding region for the 3Aprotein (4855-5124), coding region for the 3B protein (5125-5190),coding region for the 3C protein (5191-5823), coding region for the 3Dprotein (5824-7209), and the 3′UTR region including the poly(A) tail(7210-7310). The invention also provides isolated nucleic acids that areportions of the specified portions listed above. The invention alsoprovides mutants or derivatives of such isolated portions. The isolatedportions of SEQ ID NOS:1 and 168 can be subcloned into expressionvectors such that polypeptides encoded by these portions can beisolated. Further encompassed by the invention are isolated nucleicacids that can hybridize to SEQ ID NO:1 or SEQ ID NO:168, or any portionthereof, under high, moderate or low stringency conditions. Thefollowing table lists the nucleotides of SEQ ID NO:168 that encode theSVV proteins. The invention provides isolated (or purified) SVV proteinsor portions thereof. The table also lists the amino acid sequences ofthe SVV proteins with respect to the polyprotein sequence listed in SEQID NO:169.

TABLE A SVV Genome and Protein Features SVV Location in SEQ ID featureLocation in SEQ ID NO: 168 NO: 169 5′UTR 1-666 N/A (not allowed) Leader667-903 (coding sequence for Leader  1-79 peptide) VP4 904-1116 (codingsequence for VP4)  80-150 VP2 1117-1968 (coding sequence for VP2)151-434 VP3 1969-2685 (coding sequence for VP3) 435-673 VP1 2686-3474 or3477 (coding sequence for 674-936 or 937 VP1) 2A 3478-3504 (codingsequence for 2A) 938-946 2B 3505-3888 (coding sequence for 2B)  947-10742C 3889-4854 (coding sequence for 2C) 1075-1396 3A 4855-5124 (codingsequence for 3A) 1397-1486 3B 5125-5190 (coding sequence for 3B)1487-1508 3C 5191-5823 (coding sequence for 3C) 1509-1719 3D 5824-7209(coding sequence for 3D) 1720-2181 3′UTR 7210-7310 N/A

The invention provides an isolated SVV leader sequence peptide with theamino acid sequence of residues 1-79 of SEQ ID NO:169, which is encodedby nucleotides 667-903 of SEQ ID NO:168.

The invention provides an isolated SVV VP4 (1A) protein with the aminoacid sequence of residues 80-150 of SEQ ID NO:169, which is encoded bynucleotides 904-1116 of SEQ ID NO:168.

The invention provides an isolated SVV VP2 (1B) protein with the aminoacid sequence of residues 151-434 of SEQ ID NO:169, which is encoded bynucleotides 1117-1968 of SEQ ID NO:168. The invention also provides anisolated partial SVV VP2 (1B) protein with the amino acid sequence ofSEQ ID NO:4, as listed in FIG. 9 (which corresponds to amino acids 2-143of SEQ ID NO:2). The amino acid sequence of the partial SVV VP2 proteinis encoded by the nucleic acid sequence of SEQ ID NO:3, as listed inFIG. 8 (which corresponds to nucleotides 4-429 of SEQ ID NO:1).

The invention provides an isolated SVV VP3 (1C) protein with the aminoacid sequence of residues 435-673 of SEQ ID NO:169, which is encoded bynucleotides 1969-2685 of SEQ ID NO:168. The invention also provides anisolated SVV VP3 (1C) protein with the amino acid sequence of SEQ IDNO:6, as listed in FIG. 11 (which corresponds to amino acids 144-382 ofSEQ ID NO:2). The amino acid sequence of the SVV VP3 protein is encodedby the nucleic acid sequence of SEQ ID NO:5, as listed in FIG. 10 (whichcorresponds to nucleotides 430-1146 of SEQ ID NO:1).

The invention provides an isolated SVV VP1 (1D) protein with the aminoacid sequence of residues 674-937 of SEQ ID NO:169, which is encoded bynucleotides 2686-3477 of SEQ ID NO:168. The invention also provides anisolated SVV VP1 (1D) protein with the amino acid sequence of SEQ IDNO:8, as listed in FIG. 13 (which corresponds to amino acids 383-641 ofSEQ ID NO:2). The amino acid sequence of the SVV VP1 protein is encodedby the nucleic acid sequence of SEQ ID NO:7, as listed in FIG. 12 (whichcorresponds to nucleotides 1147-1923 of SEQ ID NO:1).

The invention provides an isolated SVV 2A protein with the amino acidsequence of residues 938-946 of SEQ ID NO:169, which is encoded bynucleotides 3478-3504 of SEQ ID NO:168. The invention also provides anisolated SVV 2A protein with the amino acid sequence of SEQ ID NO:10, aslisted in FIG. 15 (which corresponds to amino acids 642-655 of SEQ IDNO:2). The amino acid sequence of the SVV 2A protein is encoded by thenucleic acid sequence of SEQ ID NO:9, as listed in FIG. 14 (whichcorresponds to nucleotides 1924-1965 of SEQ ID NO:1).

The invention provides an isolated SVV 2B protein with the amino acidsequence of residues 947-1074 of SEQ ID NO:169, which is encoded bynucleotides 3505-3888 of SEQ ID NO:168. The present invention alsoprovides an isolated SVV 2B protein with the amino acid sequence of SEQID NO:12, as listed in FIG. 17 (which corresponds to amino acids 656-783of SEQ ID NO:2). The amino acid sequence of the SVV 2B protein isencoded by the nucleic acid sequence of SEQ ID NO:11, as listed in FIG.16 (which corresponds to nucleotides 1966-2349 of SEQ ID NO:1).

The invention provides an isolated SVV 2C protein with the amino acidsequence of residues 1075-1396 of SEQ ID NO:169, which is encoded bynucleotides 3889-4854 of SEQ ID NO:168. The invention also provides anisolated SVV 2C protein with the amino acid sequence of SEQ ID NO:14, aslisted in FIG. 19 (which corresponds to amino acids 784-1105 of SEQ IDNO:2). The amino acid sequence of the SVV 2B protein is encoded by thenucleic acid sequence of SEQ ID NO:13, as listed in FIG. 18 (whichcorresponds to nucleotides 2350-3315 of SEQ ID NO:1).

The invention provides an isolated SVV 3A protein with the amino acidsequence of residues 1397-1486 of SEQ ID NO:169, which is encoded bynucleotides 4855-5124 of SEQ ID NO:168. The invention also provides anisolated SVV 3A protein with the amino acid sequence of SEQ ID NO:16, aslisted in FIG. 21 (which corresponds to amino acids 1106-1195 of SEQ IDNO:2). The amino acid sequence of the SVV 3A protein is encoded by thenucleic acid sequence of SEQ ID NO:15, as listed in FIG. 20 (whichcorresponds to nucleotides 3316-3585 of SEQ ID NO:1).

The invention provides an isolated SVV 3B (VPg) protein with the aminoacid sequence of residues 1487-1508 of SEQ ID NO:169, which is encodedby nucleotides 5125-5190 of SEQ ID NO:168. The invention also providesan isolated SVV 3B protein with the amino acid sequence of SEQ ID NO:18,as listed in FIG. 23 (which corresponds to amino acids 1196-1217 of SEQID NO:2). The amino acid sequence of the SVV 3B protein is encoded bythe nucleic acid sequence of SEQ ID NO:17, as listed in FIG. 22 (whichcorresponds to nucleotides 3586-3651 of SEQ ID NO:1).

The invention provides an isolated SVV 3C (“pro” or “protease”) proteinwith the amino acid sequence of residues 1509-1719 of SEQ ID NO:169,which is encoded by nucleotides 5191-5823 of SEQ ID NO:168. Theinvention also provides an isolated SVV 3C protein with the amino acidsequence of SEQ ID NO:20, as listed in FIG. 25 (which corresponds toamino acids 1218-1428 of SEQ ID NO:2). The amino acid sequence of theSVV 3C protein is encoded by the nucleic acid sequence of SEQ ID NO:19,as listed in FIG. 24 (which corresponds to nucleotides 3652-4284 of SEQID NO:1).

The invention provides an isolated SVV 3D (“pol” or “polymerase”)protein with the amino acid sequence of residues 1720-2181 of SEQ IDNO:169, which is encoded by nucleotides 5824-7209 of SEQ ID NO:168. Theinvention also provides an isolated SVV 3D protein with the amino acidsequence of SEQ ID NO:22, as listed in FIG. 27 (which corresponds toamino acids 1429-1890 of SEQ ID NO:2). The amino acid sequence of theSVV 3C protein is encoded by the nucleic acid sequence of SEQ ID NO:19,as listed in FIG. 24 (which corresponds to nucleotides 4285-5673 of SEQID NO:1; nucleotides 5671-5673, “tga,” code for a stop-codon, which isdepicted in the amino acid sequence listings as an asterisk “*”).

The nucleic acids of the present invention include both RNA and DNAforms, and implicitly, the complementary sequences of the providedlistings.

Thus, the isolated SVV nucleic acid depicted by SEQ ID NO:168 has alength of 7,310 nucleotides that encodes a polyprotein with the aminoacid sequence depicted by SEQ ID NO:169. The isolated SVV nucleic aciddepicted by SEQ ID NO:1 has a length of 5,752 nucleotides that encodes apolypeptide with the amino acid sequence depicted by SEQ ID NO:2. TheSVV genomic sequence is translated as a single polyprotein that iscleaved into various downstream “translation products.” The presentinvention encompasses all nucleic acid fragments of SEQ ID NO: 168 andSEQ ID NO:1, and all polypeptides encoded by such fragments.

The full-length SVV polyprotein amino acid sequence is depicted by SEQID NO:169 and is encoded by nucleotides 667-7209 of SEQ ID NO:168. Themajority of the full-length SVV polyprotein amino acid sequence isencoded by nucleotides 1-5673 of SEQ ID NO:1. The polyprotein is cleavedinto three precursor proteins, P1, P2 and P3 (see FIG. 4B). P1, P2 andP3 are further cleaved into smaller products. The cleavage products ofthe structural region P1 (1ABCD; or the capsid region) are 1ABC, VP0,VP4, VP2, VP3 and VP 1. The cleavage products of the non-structuralprotein P2 (2ABC) are 2A, 2BC, 2B and 2C. The cleavage products of thenon-structural region P3 polyprotein (3ABCD) are 3AB, 3CD, 3A, 3C, 3D,3C′, and 3D′.

In certain embodiments, the invention provides isolated nucleic acidsthat comprise: (i) the coding sequence of 1ABCD or the capsid region(nucleotides 904-3477 of SEQ ID NO:168); (ii) the coding sequence of1ABC (nucleotides 904-2685 of SEQ ID NO:168); (iii) the coding sequenceof VP0 (nucleotides 904-1968 of SEQ ID NO:168); (iv) the coding sequenceof 2ABC (nucleotides 3478-4854 of SEQ ID NO:168; nucleotides 1924-3315of SEQ ID NO:1); (v) the coding sequence of 2BC (nucleotides 3505-4854of SEQ ID NO:168; nucleotides 1966-3315 of SEQ ID NO:1); (iii) thecoding sequence of 3ABCD (nucleotides 4855-7209 of SEQ ID NO:168;nucleotides 3316-5673 of SEQ ID NO:1); (iv) the coding sequence of 3AB(nucleotides 4855-5190 of SEQ ID NO:168; nucleotides 3316-3651 of SEQ IDNO:1); and (v) the coding sequence of 3CD (nucleotides 5191-7209 of SEQID NO:168; nucleotides 3652-5673 of SEQ ID NO:1). The invention alsoprovides isolated proteins or peptides encoded by the coding sequencesdescribed above, including fragments thereof.

The basic capsid structure of picornaviruses consists of a denselypacked icosahedral arrangement of 60 protomers, each consisting of 4polypeptides, VP1, VP2, VP3 and VP4, all of which are derived from thecleavage of the original protomer, VP0. The SVV virus particle is about27 nm in diameter (see FIG. 2), which is consistent with the size ofother picornavirus particles, which are about 27-30 nm in diameter.

The kinetics of picornavirus replication is rapid, the cycle beingcompleted in about 5-10 hours (typically by about 8 hours) (see FIG. 68for a schematic of the picornavirus replication cycle). Upon receptorbinding, the genomic RNA is released from the particle into thecytoplasm. Genomic RNA is then translated directly by polysomes, but inabout 30 minutes after infection, cellular protein synthesis declinessharply, almost to zero. This phenomenon is called “shutoff,” and is aprimary cause of cytopathic effects (cpe). Shutoff appears to be due tocleavage of the host cell's 220 kDa cap-binding complex (CBC) that isinvolved in binding the m7G cap structure at the 5′ end of alleukaryotic mRNA during initiation of translation. The cleavage of theCBC appears to be caused by the 2A protease.

The 5′ UTR contains the IRES. Normally, eukaryotic translation isinitiated when ribosomes bind to the 5′ methylated cap and then scansalong the mRNA to find the first AUG initiation codon. The IRESovercomes this process and allows Picornavirus RNA's to continue to betranslated after degradation of CBC. In one embodiment, the inventionprovides for an isolated nucleic acid comprising the SVV IRES, whereinthe IRES is contained within the 5′UTR. In one embodiment, the SVV IREScan be from nucleotides 300-366 of SEQ ID NO:168. The 5′UTR of SVV ispresent at nucleotides 1-666 of SEQ ID NO:168.

The virus polyprotein is initially cleaved by the 2A protease intopolyproteins P1, P2 and P3 (see FIG. 4B). Further cleavage events arethen carried out by 3C, the main picornavirus protease. One of thecleavage products made by 3C is the virus RNA-dependent RNA polymerase(3D), which copies the genomic RNA to produce a negative (−) sensestrand. The (−) sense strand forms the template for the (+) strand(genomic) RNA synthesis. Some of the (+) strands are translated toproduce additional viral proteins are some (+) strands are packaged intocapsids to form new virus particles.

The (+) strand RNA genome is believed to be packaged into preformedcapsids, although the molecular interactions between the genome and thecapsid are not clear. Empty capsids are common in all picornavirusinfections. The capsid is assembled by cleavage of the P1 polyproteinprecursor into a protomer consisting of VP0, VP3, and VP1, which jointogether enclosing the genome. Maturation and infectivity of the virusparticle relies on an internal autocatalytic cleavage of VP0 into VP2and VP4. Release of newly formed virus particles occurs when the celllyses.

The present invention also provides an isolated virus having all theidentifying characteristics and nucleic acid sequence of ATCC PatentDeposit number PTA-5343. Viruses of the present invention can bedirected to the PTA-5343 isolate, variants, homologues, derivatives andmutants of the PTA-5343 isolate, and variants, homologues, derivativesand mutants of other picornaviruses that are modified in respect tosequences of SVV (both wild-type as disclosed herein and mutant) thatare determined to be responsible for its oncolytic properties.

The present invention further provides antibodies that are specificagainst: the isolated SVV having the ATTC Patent Deposit numberPTA-5343, and epitopes from the isolated SVV proteins having the aminoacid sequences SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and169 (including entire polyprotein, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A,3B, 3C, 3D, and portions thereof; see Table A supra for amino acids inSEQ ID NO:169 that make up these proteins). The invention also includesantibodies that are specific against epitopes from the proteins that areencoded by fragments or portions of SEQ ID NO:1 or SEQ ID NO:168.

Comparative analyses of the RNA sequences from a variety of cardiovirusisolates have shown >45% nucleotide identity between genomes.Cardioviruses can be subclassified into the EMC-like viruses(“EMCV”—such as, Mengo, B, R; and also MM, ME, Columbia-SK), theTheiler's-like viruses (“TMEV”—such as, BeAn, DA and GD VII strains),and the Vilyuisk viruses.

In analyzing the SVV sequence to other viruses, it appears that SVV is acardiovirus (see Example 4 and Figures referenced therein). If EMCV andTMEV are taken as the standard cardioviruses, SVV is clearly not atypical cardiovirus. However, even these two viruses have theirdifferences, notably in the 5′ UTR (Pevear et al., 1987, J. Virol., 61:1507-1516). Phylogenetically SVV clusters with EMCV and TMEV in much ofits polyprotein (P1, 2C, 3C^(pro) and 3D^(pol) regions; see FIGS.31-37), indicating that SVV is most likely a cardiovirus.

SVV is phylogenetically similar to cardioviruses, but it has now beendetermined to be in a separate tree (see FIG. 86). SVV can be in aseparate genus because: (1) SVV IRES is Type IV (cardiovirus IRES areType II); (2) multiple unique viruses (“SVV-like picornaviruses”) aremore similar to SVV than SVV is to other cardioviruses (see Example 18and FIGS. 87-89); and antibodies that can neutralize SVV infection ofpermissive cell lines or were raised against SVV are able to bind to theSVV-like picornaviruses. Thus, an SVV-like picornavirus can be used inany of the present methods, including the methods to treat cancer, whereit is determined that the SVV-like picornavirus is naturally oncolyticor is made to be oncolytic (for example, by designing mutations in theSVV-like picornavirus genome based on the SVV sequence). In oneembodiment, MN 88-36696 is used in the present methods to treat cancer.

Methods for Treating Cancer:

The present invention provides methods for cancer therapy using virusesmodified in view of the oncolytic properties of SVV, includingpicornaviruses (including SVV-like picornaviruses), derivatives,variants, mutants or homologues thereof. The present invention showsthat wild-type SVV (i.e., ATTC Patent Deposit number PTA-5343) has theability to selectively kill some types of tumors. For example, SVV canselectively kill tumor cells that have neurotropic or neuroendocrineproperties, including small cell lung cancer (SCLC) and neuroblastomas.Other examples of neuroendocrine tumors that are contemplated to betreated by the viruses of the present invention include, but are notlimited to: adrenal pheochromocytomas, gastrinomas (causingZollinger-Ellison syndrome), glucagonomas, insulinomas, medullarycarcinomas (including medullary thyroid carcinoma), multiple endocrineneoplasia syndromes, pancreatic endocrine tumors, paragangliomas,VIPomas (vasoactive intestinal polypeptide tumor), islet cell tumors,and pheochromocytoma.

In one embodiment, the invention provides methods for treating orreducing neuroendocrine tumors by administering to a subject SVV or anSVV-like picornavirus, where the neuroendocrine tumor expresses (oroverexpresses) one or more neuroendocrine tumor markers, including butnot limited to, NTR (Neurotensin receptor), ATOH (, GL11, Myc, GRPreceptors, GRP, Neuronal enolase (neuron specific enolase (NSE)),carcinoembryonic antigen (CEA), chromoganin A, NCAM, IgF2, BCL-2, sonichedgehog pathway, and a chemokine receptor.

Also encompassed in the present invention are the four types ofneuroendocrine lung tumors. The most serious type, small cell lungcancer (SCLC), is among the most rapidly growing and spreading of allcancers. Large cell neuroendocrine carcinoma is a rare cancer that, withthe exception of the size of the cells forming the cancer, is verysimilar to SCLC in its prognosis and in how patients are treated.Carcinoid tumors, also known as carcinoids, comprise the other 2 typesof lung neuroendocrine cancer. These two types are typical carcinoid andatypical carcinoid.

Not being bound by theory, the ability of SVV to specifically kill tumorcells may include, but is not limited to: selective replication, cellprotein synthesis shut-off, apoptosis, lysis via tumor-selective cellentry, tumor-selective translation, tumor-selective proteolysis,tumor-selective RNA replication, and combinations thereof.

SVV has many advantageous characteristics over other oncolytic viruses,including modified adenoviruses, for example: (i) SVV has a very highselectivity for cancers with neural properties, including SCLC, Wilms'tumor, retinoblastoma, and neuroblastoma—for example, SVV shows agreater than 10,000-fold selectivity toward neuroendocrine tumor cells;(ii) SVV has been shown to have a 1,000 fold better cell-killingspecificity than chemotherapy treatments; (iii) SVV exhibits no overttoxicity in mice following systemic administration with as high as 10¹⁴viral particles per kilogram; (iv) the efficacy of SVV is very robust inthat 100% of large pre-established tumors can be eradicated in mice,with no recurrence of tumor growth; (v) SVV can be purified to hightiter and can be produced at more than 200,000 particles per cell inpermissive cell lines; (vi) SVV has a small size (the SVV virus particleis less than 30 nm in diameter) enabling better penetration and spreadin tumors than other oncolytic viruses, (vii) SVV replicates quickly(less than 12 hours) and (viii) no modification of SVV is necessary forits use as a specific anti-tumor agent.

Further, initial studies (see Example 6) indicate some additionalfactors that make SVV an advantageous tool for oncolytic viral therapy:(i) human serum samples do not contain neutralizing antibodies directedagainst SVV; (ii) SVV is not inhibited by complement; and (iii) SVV doesnot produce hemagglutination of human erythrocytes. All of these factorscontribute to the fact that SVV exhibits a longer circulation time invivo than other oncolytic viruses (for example, see Example 7).

The present invention provides methods for selectively killing aneoplastic cell in a cell population that comprises contacting aneffective amount of SVV with said cell population under conditions wherethe virus can transduce the neoplastic cells in the cell population,replicate and kill the neoplastic cells. Besides methods where SVV killstumor cells in vivo, the present methods encompass embodiments where thetumors can be: (1) cultured in vitro when infected by SVV; (2) culturedin the presence of non-tumor cells; and (3) the cells are mammalian(both tumor and non-tumor cells), including where the cells are humancells. The in vitro culturing of cells and infection by SVV can havevarious applications. For example, in vitro infection be used as amethod to produce large amounts of SVV, as method for determining ordetecting whether neoplastic cells are present in a cell population, oras a method for screening whether a mutant SVV can specifically targetand kill various tumor cell or tissue types.

The present invention further provides an ex vivo method of treatingcancer wherein cells are isolated from a human cancer patient, culturedin vitro, infected with a SVV which selectively kills the cancer cells,and the non-tumor cells are introduced back to the patient.Alternatively, cells isolated form a patient can be infected with SVVand immediately introduced back to the patient as a method foradministering SVV to a patient. In one embodiment, the cancer cells areof a hematopoietic origin. Optionally, the patient may receive treatment(e.g., chemotherapy or radiation) to destroy the patient's tumor cell invivo before the cultured cells are introduced back to the patient. Inone embodiment, the treatment may be used to destroy the patient's bonemarrow cells.

Polymer coated SVV can be used to target the SVV to any specific celltype. This coating strategy can also be used to overcome antibodies toSVV.

SVV possesses potent antitumor activity against tumor cell-types withneural characteristics. SVV does not exhibit cytolytic activity againsttested normal human. Further SVV is not cytotoxic to primary humanhepatocytes. Table 1 below summarizes initial studies that have beenconducted to determine the in vitro cytolytic potency of SVV againstselected tumor cell types.

TABLE 1 SVV Cytolytic Potency Against Selected Tumor Cell-Types CellLine Cell Type EC₅₀ (VP/cell) H446 Human SCLC 0.0012 PER.C6 HumanEmbryonic Retinoblast 0.02 H69AR SCLC-Multidrug Resistant 0.035 293 AD5DNA Transformed Human 0.036 Kidney Y79 Human Retinoblastoma 0.00035IMR32 Human Brain Neuroblastoma 0.035 D283 Med Human Brain Cerebellar0.25 Medulloblastoma SK-N-AS Human Brain Neuroblastoma 0.474 N1E-115Mouse Neuroblastoma 0.0028 BEKPCB3E1 Bovine embryonic Kidney cells 0.99transformed with Ad5E1 H1299 Human non-SCLC 7.66 ST Porcine Testis 5.9DMS153 Human SCLC 9.2 BEK Bovine Embryonic Kidney 17.55 M059K HumanBrain Malignant 1,061 Glioblastoma PK15 Porcine Kidney 1,144 FBRC FetalBovine Retina 10,170 HCN-1A Human Brain 23,708 H460 Human LCLC >30,000(inactive) Neuro 2A Mouse Neuroblastoma >30,000 (inactive) DMS79 HumanSCLC >30,000 (inactive) H69 Human SCLC >30,000 (inactive) C8D30 MouseBrain >30,000 (inactive) MRC-5 Human Fetal Lung Fibroblast >30,000(inactive) HMVEC Neonatal vascular endothelial cells >30,000 (inactive)HMVEC Adult vascular endothelial cells >30,000 (inactive) A375-S2 HumanMelanoma >30,000 (inactive) SK-MEL-28 Melanoma >30,000 (inactive) PC3Human prostate cancer >30,000 (inactive) PC3M2AC6 Human prostatecancer >30,000 (inactive) LnCap Human Prostate cancer >30,000 (inactive)DU145 Human prostate cancer >30,000 (inactive)

Table 1-A below provides a list of cell lines that are permissive arenon-permissive to SVV infection. The Table shows the cytolytic potencyand selectivity of SVV.

TABLE 1 In Vitro Cytolytic Potency and Selectivity of SVV Cell LineSpecies Stage State Organ Type Metastatic Site EC50* PERMISSIVE Y79Human Adult Cancer Eye, Retina Retinoblastoma 0.00035, 0.0007 NCI-H446Human Adult Metastatic Lung Variant Small Cell Pleural effusion 0.0012,0.002, Cancer Lung Carcinoma 0.0007 (SCLC) N1E-115 Murine Adult CancerBrain Neuroblastoma 0.0028, 0.001 NCI-H1770 Human Adult Metastatic LungNon-Small Cell Lymph Node 0.00724 Cancer Lung Carcinoma (NSCLC) NCI-H82Human Adult Metastatic Lung Variant Small Cell Pleural effusion 0.015Cancer Lung Carcinoma (SCLC) PER.C6 ® Human Fetal Cancer Eye, RetinaRetinoblast 0.02, 0.0049 NCI-H69AR Human Adult Cancer Lung Small CellLung 0.035, 0.05 Carcinoma, multi-drug resistant (SCLC) SK-NEP-1 HumanAdult Metastatic Kidney Wilms' Tumor Pleural effusion 0.03 Cancer IMR-32Human Adult Cancer Brain Neuroblastoma 0.035, 0.0059, 0.05 NCI-H187Human Adult Metastatic Lung Classic Small Cell Pleural effusion 0.00343Cancer Lung Carcinoma (SCLC) NCI-H209 Human Adult Metastatic Lung SmallCell Lung Bone Marrow 0.04 Cancer Carcinoma (SCLC) NCI-H1184 Human AdultMetastatic Lung Small Cell Lung Lymph Node 0.155 Cancer Carcinoma (SCLC)D283 Med Human Adult Metastatic Brain, Medulloblastoma Peritoneum 0.25Cancer Cerebellum SK-N-AS Human Adult Metastatic Brain NeuroblastomaBone Marrow 0.474 Cancer BEK PCB3E1 Bovine Fetal Normal, Ad5 KidneyAd5E1 transformed 0.99 transformed ST Porcine Fetal Normal, Testis 5.9immortalized NCI-H1299 Human Adult Metastatic Lung Large Cell Lung LymphNode 7.66, 4.8 Cancer Carcinoma DMS 153 Human Adult Metastatic LungSmall Cell Lung Liver 9.2 Cancer Carcinoma (SCLC) NCI-H295R Human AdultCancer Adrenal Gland, Adrenocortical 16.5 Cortex Carcinoma BEK BovineFetal Normal, Kidney 17.55 immortalized PPASMC Porcine Adult Normal,Lung, Smooth Muscle Cells 18.4 Primary Pulmonary Artery PCASMC PorcineAdult Normal, Heart, Coronary Smooth Muscle Cells 11.9 Primary ArteryPAoSMC Porcine Adult Normal, Heart, Aorta Smooth Muscle Cells 88 PrimaryNCI-H526 Human Adult Metastatic Lung Variant Small Cell Bone Marrow 46.4Cancer Lung Carcinoma (SCLC) OVCAR-3 Human Adult Cancer OvaryAdenocarcinoma 39 ESK-4 Porcine Fetal Normal, Kidney Fibroblast 60Immortalized SW-13 Human Adult Cancer Adrenal Gland, Small Cell <100Cortex Adenocarcinoma 293 Human Fetal Normal, Ad5 Kidney Ad5 transformed0.036, 184.8 transformed Hs 578T Human Adult Cancer Breast Carcinoma 273Hs 1.Tes Human Fetal Normal, Testis 416 Immortalized LOX IMVI HumanAdult Cancer Skin Melanoma 569 PK(15) Porcine Adult Normal, Kidney 1144,129 Immortalized NON PERMISSIVE WI-38 Human Fetal Normal, LungFibroblast >10,000 Immortalized IMR-90 Human Fetal Normal, LungFibroblast >10,000 Immortalized MRC-5 Human Fetal Normal, LungFibroblast >10,000 Immortalized HCN-1A Human Adult Normal, Brain,Cortical >10,000 Immortalized Neuron HMVEC Human Adult Normal, SkinMicrovascular >10,000 (neonatal) Primary Endothelial Cells HMVEC HumanAdult Normal, Skin Microvascular >10,000 Primary Endothelial Cells HUVECHuman Adult Normal, Umbilical Vein Endothelial Cells >10,000 Primary HREHuman Adult Normal, Kidney Epithelial Cells >10,000 Primary HRCE HumanAdult Normal, Kidney Cortical Epithelial Cells >10,000 Primary PHH HumanAdult Normal, Liver Hepatocyte >10,000 Primary HCASMC-c Human AdultNormal, Heart, Coronary Smooth Muscle Cells >10,000 Primary Artery HCAECHuman Adult Normal, Heart, Coronary Endothelial Cells >10,000 PrimaryArtery HAEC Human Adult Normal, Heart, Aorta Endothelial Cells >10,000Primary HAoSMC-c Human Adult Normal, Heart, Aorta Smooth MuscleCells >10,000 Primary NHA Human Adult Normal, Brain Astrocytes 1713Primary HPASMC Human Adult Normal, Lung Smooth Muscle Cells >10,000Primary PBMC Human Adult Normal, Peripheral Blood MononuclearCells >10,000 Primary SF-295 Human Adult Cancer BrainGlioblastoma >10,000 U251 Human Adult Cancer Brain Glioblastoma >10,000SF-539 Human Adult Cancer Brain Glioblastoma >10,000 SNB-19 Human AdultCancer Brain Glioblastoma >10,000 SF-268 Human Adult Cancer BrainGlioblastoma 3103 U-118MG Human Adult Cancer Brain Glioblastoma, >10,000Astrocytoma SNB-75 Human Adult Cancer Brain Astrocytoma >10,000 M059KHuman Adult Cancer Brain, Glial Cell Malignant 1061 Glioblastoma KKHuman Adult Cancer Brain, Glial Cell Glioblastoma >10,000 HCC-2998 HumanAdult Cancer Colon Carcinoma >10,000 KM12 Human Adult Cancer ColonCarcinoma >10,000 HT-29 Human Adult Cancer Colon Adenocarcinoma >10,000HCT 116 Human Adult Cancer Colon Carcinoma >10,000 HCT-15 Human AdultCancer Colon Carcinoma >10,000 COLO 205 Human Adult Metastatic ColonAdenocarcinoma Ascites >10,000 Cancer SW620 Human Adult Metastatic ColonColorectal Carcinoma Lymph Node 6503, >10,000 Cancer PC3M-2AC6 HumanAdult Cancer Prostate >10,000 PC3M-2AC6 + Human Adult Cancer Prostate ND2-AP PC-3 Human Adult Metastatic Prostate Adenocarcinoma Bone >10,000Cancer LNCaP.FGC Human Adult Metastatic Prostate Adenocarcinoma LymphNode >10,000 Cancer DU 145 Human Adult Metastatic ProstateAdenocarcinoma Brain >10,000 Cancer Hep3B Human Adult Cancer LiverHepatocellular >10,000 Carcinoma Hep G2 Human Adult Cancer LiverHepatocellular >10,000 Carcinoma 786-O Human Adult Cancer Kidney ClearCell >10,000 Adenocarcinoma TK-10 Human Adult Cancer KidneyCarcinoma >10,000 RXF 393 Human Adult Cancer Kidney Carcinoma >10,000UO-31 Human Adult Cancer Kidney Carcinoma >10,000 SN12C Human AdultCancer Kidney Carcinoma >10,000 A-498 Human Adult Cancer KidneyCarcinoma >10,000 ACHN Human Adult Cancer Kidney Carcinoma >10,000 SW839Human Adult Cancer Kidney Renal Clear Cell >10,000 Adenocarcinoma Caki-1Human Adult Metastatic Kidney Clear Cell Skin >10,000 CancerAdenocarcinoma 5637 Human Adult Cancer Bladder Carcinoma >10,000NCI-H1339 Human Adult Cancer Lung >10,000 NCI-H1514 Human Adult CancerLung >10,000 A549 Human Adult Cancer Lung Carcinoma >10,000 S8 HumanAdult Cancer Lung Carcinoma >10,000 NCI-H727 Human Adult Cancer LungCarcinoid >10,000 NCI-H835 Human Adult Cancer Lung Carcinoid >10,000UMC-11 Human Adult Cancer Lung Carcinoid >10,000 DMS 114 Human AdultCancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) DMS 53 Human AdultCancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) NCI-H69 Human AdultCancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) NCI-H2195 HumanAdult Metastatic Lung Small Cell Lung Bone Marrow >10,000 CancerCarcinoma (SCLC) DMS 79 Human Adult Metastatic Lung Small Cell LungPleural effusion >10,000 Cancer Carcinoma (SCLC) NCI-H146 Human AdultMetastatic Lung Classic Small Cell Bone Marrow >10,000 Cancer LungCarcinoma (SCLC) NCI-H1618 Human Adult Metastatic Lung Classic SmallCell Bone Marrow >10,000 Cancer Lung Carcinoma (SCLC) NCI-H345 HumanAdult Metastatic Lung Classic Small Cell Bone Marrow >10,000 Cancer LungCarcinoma (SCLC) HOP-62 Human Adult Cancer Lung Non-Small Cell >10,000Lung Carcinoma (NSCLC) EKVX Human Adult Cancer Lung Non-SmallCell >10,000 Lung Carcinoma (NSCLC) HOP-92 Human Adult Cancer LungNon-Small Cell >10,000 Lung Carcinoma (NSCLC) NCI-H522 Human AdultCancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) NCI-H23 HumanAdult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC)NCI-H322M Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma(NSCLC) NCI-H226 Human Adult Metastatic Lung Squamous Cell Pleuraleffusion >10,000 Cancer Carcinoma, Mesothelioma (NSCLC) NCI-H460 HumanAdult Metastatic Lung Large Cell Lung Pleural effusion >10,000 CancerCarcinoma HeLa, HeLa Human Adult Cancer Cervix Adenocarcinoma >10,000 S3CCRF-CEM Human Adult Cancer Peripheral Acute Lymphoblastic >10,000Blood, T Leukemia (ALL) lymphoblast MOLT-4 Human Adult Cancer PeripheralAcute Lymphoblastic >10,000 Blood, T Leukemia (ALL) lymphoblast RPMI8226 Human Adult Cancer Peripheral Plasmacytoma, >10,000 Blood, BMyeloma lymphocyte SR Human Adult Metastatic Lymphoblast Large CellPleural effusion >10,000 Cancer Lymphoblastic Lymphoma HL-60(TB) HumanAdult Cancer Peripheral Acute Promyelocytic >10,000 Blood, Leukemia(APL) Promyleoblast K-562 Human Adult Metastatic Bone Marrow ChronicMyelogenous Pleural effusion >10,000 Cancer Leukemia (CML) UACC-257Human Adult Cancer Skin Melanoma >10,000 M14 Human Adult Cancer SkinMelanoma >10,000 UACC-62 Human Adult Cancer Skin Melanoma 6614 SK-MEL-2Human Adult Cancer Skin Malignant Melanoma >10,000 SK-MEL-28 Human AdultCancer Skin Malignant Melanoma >10,000 A375.S2 Human Adult Cancer SkinMalignant Melanoma >10,000 SK-MEL-28 Human Adult Cancer Skin MalignantMelanoma >10,000 SK-MEL-5 Human Adult Metastatic Skin Malignant MelanomaLymph Node >10,000 Cancer MALME-3M Human Adult Metastatic Skin MalignantMelanoma Lung >10,000 Cancer BT-549 Human Adult Cancer Breast DuctalCarcinoma >10,000 NCI/ Human Adult Cancer Breast Carcinoma >10,000ADR-RES MCF7 Human Adult Metastatic Breast Adenocarcinoma Pleuraleffusion >10,000 Cancer MDA-MB-231 Human Adult Metastatic BreastAdenocarcinoma Pleural effusion >10,000 Cancer T-47D Human AdultMetastatic Breast Ductal Carcinoma Pleural effusion >10,000 CancerMDA-MB-435 Human Adult Metastatic Breast Ductal Pleural effusion >10,000Cancer Adenocarcinoma IGR-OV1 Human Adult Cancer Ovary Carcinoma >10,000OVCAR-4 Human Adult Cancer Ovary Adenocarcinoma >10,000 OVCAR-5 HumanAdult Cancer Ovary Adenocarcinoma >10,000 OVCAR-8 Human Adult CancerOvary Adenocarcinoma >10,000 SK-OV-3 Human Adult Metastatic OvaryAdenocarcinoma Ascites >10,000 Cancer BxPC-3 Human Adult Cancer PancreasAdenocarcinoma >10,000 AsPC-1 Human Adult Metastatic PancreasAdenocarcinoma Ascites >1000 Cancer NCI-H295 Human Adult Cancer AdrenalGland, Adrenocortical >10,000 Cortex Carcinoma TT Human Adult CancerThyroid Medullary Carcinoma >10,000 C8-D30 Murine Adult NormalBrain, >10,000 Cerebellum LLC1 Murine Adult Cancer Lung Lewis LungCarcinoma >10,000 RM-1 Murine Adult Cancer Prostate >10,000 MLTC-1Murine Adult Cancer Testis Leydig Cell Tumor >10,000 KLN 205 MurineAdult Cancer Lung Squamous Cell >10,000 Carcinoma CMT-64 Murine AdultCancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) CMT-93 Murine AdultCancer Rectum Polyploid Carcinoma >10,000 B16-F0 Murine Adult CancerSkin Melanoma >10,000 RM-2 Murine Adult Cancer Prostate >10,000 RM-9Murine Adult Cancer Prostate >10,000 Neuro-2A Murine Adult Cancer BrainNeuroblastoma >10,000 FBRC Bovine Fetal Eye, Retina >10,000 MDBK BovineAdult Normal, Kidney >10,000 Immortalized CSL 503 Ovine Adult Normal,Lung Ad5E1 >10,000 Immortalized transformed OFRC Ovine Adult Normal,Eye, Retina Ad5E1 >10,000 Immortalized transformed PC-12 Rat AdultCancer Adrenal Gland Pheochromocytoma >10,000 Vero Monkey Adult Normal,Kidney >10,000 Immortalized PAOEC Porcine Adult Normal, Heart, AortaEndothelial Cells >10,000 Primary PCAEC Porcine Adult Normal, Heart,Coronary Endothelial Cells >10,000 Primary Artery PPAEC Porcine AdultNormal, Lung, Endothelial Cells >10,000 Primary Pulmonary Artery TBDNCI-H289 Human Adult Cancer Lung TBD NCI-H1963 Human Adult Cancer LungSmall Cell Lung TBD Carcinoma (SCLC) NCI-H2227 Human Adult Cancer LungSmall Cell Lung TBD Carcinoma (SCLC) NCI-H378 Human Adult MetastaticLung Classic Small Cell Pleural effusion TBD Cancer Lung Carcinoma(SCLC) NCI-H2107 Human Adult Metastatic Lung Small Cell Lung Bone MarrowTBD Cancer Carcinoma (SCLC) HCC970 Human Adult Metastatic Lung SmallCell Lung Bone Marrow TBD Cancer Carcinoma (SCLC) HCC33 Human AdultMetastatic Lung Small Cell Lung Pleural effusion <1000/TBD CancerCarcinoma (SCLC) BON Human Adult Cancer Pancreas Carcinoid TBD H1T-T15Hamster Adult Normal, Pancreas Islets of Langerhans, TBD Immortalizedb-cell *EC50 determined after 3 days except where noted

Table 1-A lists the results of SVV permissivity experiments on 165primary cells and cell lines, representing 22 tissues from 8 differentspecies. The results indicate that virtually all adult normal arenonpermissive for SVV. Thirteen primary adult human cell cultures testedwere nonpermissive. Of the twelve bovine, ovine, porcine and primatenormal cell cultures tested, only three cell cultures were permissive,which were porcine smooth muscle cells. This result is consistent withthe hypothesis that the natural host for SVV may be pigs. Besides theporcine smooth muscle cells, only neuroendocrine cancer cell lines ormost fetal lines were permissive.

Murine studies (see Examples) show that SVV can specifically kill tumorswith great efficacy and specificity in vivo. These in vivo studies showthat SVV has a number of advantages over other oncolytic viruses. Forexample, one important factor affecting the ability of an oncolytictumor virus to eradicate established tumors is viral penetration. Instudies with adenoviral vectors, Ad5 based vectors had no effect on SCLCtumor development in athymic mice. Based on immunohistochemical results,adenovirus did not appear to penetrate the established tumors. Incontrast, SVV was able to eliminate H446 SCLC tumors in athymic nudemice following a single systemic administration. SVV has a small size(<30 nm in diameter) enabling better penetration and spread in tumortissue than other viruses, and thus, the small size of SVV maycontribute to its ability to successfully penetrate and eradicateestablished tumors.

Additional in vivo tests demonstrate the efficacy of a singleintravenous dose of SVV in murine tumor models using athymic nude miceand immunocompetent mice. The tumor models tested were: (1) H446 (humanSCLC); (2) Y79 (human retinoblastoma); (3) H69AR (human multi-drugresistant SCLC); (4) H1299 (human NSCLC); and (5) N1E-115 (murineneuroblastoma). The results of these tests are shown in FIGS. 90A-E andExample 11. The tests demonstrate efficacy of a single intravenous doseof SVV in all models and show an agreement between relative ranks of invitro ED₅₀ and in vivo efficacy in human xenograft models. The resultsin the N1E-115 immunocompetent murine neuroblastoma model shows that SVVcan be efficacious against tumors in subjects with normal immunesystems.

Chemoresistance is a major issue facing any patient that receiveschemotherapy as a facet of cancer therapy. Patients that becomechemoresistant often, if not always, have a much poorer prognosis andmay be left with no alternative therapy. It is well known that one ofthe major causes of chemoresistance is the expression, over expression,or increased activity of a family of proteins called Multiple DrugResistant proteins (MRPs). Applicants have found that a sensitivity ofcertain tumor cells for SVV is also correlated with the chemoresistantstate of cancer cells and MRP expression. H69 is a chemosensitive(adriamycin) cell line that is resistant to SVV in vitro, whereas H69ARis a chemoresistant cell line that overexpresses MRPs and is sensitiveto SVV (see Table 1). Evidence indicates that overexpression of MRPs,including MDR, correlates with sensitivity of cells to SVV killing.Thus, in one embodiment, the present invention provides a method fortreating cancer wherein SVV kills cells overexpressing an MRP.

The invention also provides methods for treating diseases that are aresult of abnormal cells, such as abnormally proliferative cells. Themethod comprises contacting said abnormal cells with SVV in a mannerthat results in the destruction of a portion or all of the abnormalcells. SVV can be used to treat a variety of diseases that are a resultof abnormal cells. Examples of these diseases include, but are notlimited to, cancers wherein the tumor cells display neuroendocrinefeatures and neurofibromatosis.

Neuroendocrine tumors can be identified by a variety of methods. Forexample, neuroendocrine tumors produce and secrete a multitude ofpeptide hormones and amines. Some of these substances cause a specificclinical syndrome: carcinoid, Zollinger-Ellison, hyperglycemic,glucagonoma and WDHA syndrome. Specific markers for these syndromes arebasal and/or stimulated levels of urinary 5-HIAA, serum or plasmagastrin, insulin, glucagon and vasoactive intestinal polypeptide,respectively. Some carcinoid tumors and about one third of endocrinepancreatic tumors do not present any clinical symptoms and are called‘nonfunctioning’ tumors. Therefore, general tumor markers such aschromogranin A, pancreatic polypeptide, serum neuron-specific enolaseand subunits of glycoprotein hormones have been used for screeningpurposes in patients without distinct clinical hormone-related symptoms.Among these general tumor markers chromogranin A, although its precisefunction is not yet established, has been shown to be a very sensitiveand specific serum marker for various types of neuroendocrine tumors.This is because it may also be elevated in many cases of lesswell-differentiated tumors of neuroendocrine origin that do not secreteknown hormones. At the moment, chromogranin A is considered the bestgeneral neuroendocrine serum or plasma marker available both fordiagnosis and therapeutic evaluation and is increased in 50-100% ofpatients with various neuroendocrine tumors. Chromogranin A serum orplasma levels reflect tumor load, and it may be an independent marker ofprognosis in patients with midgut carcinoids.

The invention also provides a pharmaceutical composition comprising SVVand a pharmaceutically acceptable carrier. Such compositions, which cancomprise an effective amount of SVV in a pharmaceutically acceptablecarrier, are suitable for local or systemic administration toindividuals in unit dosage forms, sterile parenteral solutions orsuspensions, sterile non-parenteral solutions or oral solutions orsuspensions, oil in water or water in oil emulsions, and the like.Formulations for parenteral and non-parenteral drug delivery are knownin the art. Compositions also include lyophilized and/or reconstitutedforms of SVV. Acceptable pharmaceutical carriers are, for example,saline solution, protamine sulfate (Elkins-Sinn, Inc., Chemy Hill,N.J.), water, aqueous buffers, such as phosphate buffers and Trisbuffers, or Polybrene (Sigma Chemical, St. Louis, Mo.) andphosphate-buffered saline and sucrose. The selection of a suitablepharmaceutical carrier is deemed to be apparent to those skilled in theart from the teachings contained herein. These solutions are sterile andgenerally free particulate matter other than SVV. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate, etc. Excipients that enhance infection of cells by SVV may beincluded.

SVV is administered to a host or subject in an amount that is effectiveto inhibit, prevent or destroy the growth of the tumor cells throughreplication of the virus in the tumor cells. Methods that utilize SVVfor cancer therapy include systemic, regional or local delivery of thevirus at safe, developable, and tolerable doses to elicittherapeutically useful destruction of tumor cells. Even followingsystemic administration, the therapeutic index for SVV is at least 10,preferably at least 100 or more preferably at least 1000. In general,SVV is administered in an amount of between 1×10⁸ and 1×10¹⁴ vp/kg. Theexact dosage to be administered is dependent upon a variety of factorsincluding the age, weight, and sex of the patient, and the size andseverity of the tumor being treated. The viruses may be administered oneor more times, which may be dependent upon the immune response potentialof the host. Single or multiple administrations of the compositions canbe carried out with dose levels and pattern being selected by thetreating physician. If necessary, the immune response may be diminishedby employing a variety of immunosuppressants, so as to permit repetitiveadministration and/or enhance replication by reducing the immuneresponse to the viruses. Anti-neoplastic viral therapy of the presentinvention may be combined with other anti-neoplastic protocols. Deliverycan be achieved in a variety of ways, employing liposomes, directinjection, catheters, topical application, inhalation, etc. Further, aDNA copy of the SVV genomic RNA, or portions thereof, can also be amethod of delivery, where the DNA is subsequently transcribed by cellsto produce SVV virus particles or particular SVV polypeptides.

A therapeutically effective dose refers to that amount of the virus thatresults in amelioration of symptoms or a prolongation of survival in apatient. Toxicity and therapeutic efficacy of viruses can be determinedby standard procedures in cell cultures or experimental animals, e.g.,for determining the LD₅₀ (the dose lethal to 50% of the population ofanimals or cells; for viruses, the dose is in units of vp/kg) and theED₅₀ (the dose—vp/kg—therapeutically effective in 50% of the populationof animals or cells) or the EC₅₀ (the effective concentration—vp/cell(see Table 1 for example)—in 50% of the population of animals or cells).The dose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio between LD₅₀ and ED₅₀ orEC₅₀. Viruses which exhibit high therapeutic indices are preferred. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in human. The dosage ofviruses lies preferably within a range of circulating concentrationsthat include the ED₅₀ or EC₅₀ with little or no toxicity. The dosage mayvary within this range depending upon the dosage form employed and theroute of administration utilized.

In yet another aspect, a method for treating a host organism having aneoplastic condition is provided, comprising administering atherapeutically effective amount of a viral composition of the inventionto said host organism. In one embodiment, the neoplastic tissue isabnormally proliferating, and the neoplastic tissue can be malignanttumor tissue. Preferably, the virus is distributed throughout the tissueor tumor mass due to its capacity for selective replication in the tumortissue. Neoplastic conditions potentially amenable to treatment with themethods of the invention include those with neurotropic properties.

Methods for Producing the Viruses of the Present Invention:

Methods for producing the present viruses to very high titers and yieldsare additional aspects of the invention. As stated, SVV can be purifiedto high titer and can be produced at more than 200,000 particles percell in permissive cell lines. Cells that are capable of producing highquantities of viruses include, but are not limited to, PER.C6 (Fallauxet al., Human Gene Therapy, 9:1909-1917, 1998), H446 (ATCC#HTB-171) andthe other cell lines listed in Table 1 where the EC₅₀ value is less than10.

For example, the cultivation of picornaviruses can be conducted asfollows. The virus of interest is plaque purified and a well-isolatedplaque is picked and amplified in a permissive cell line, such asPER.C6. A crude virus lysate (CVL) from the infected cells can be madeby multiple cycles of freeze and thaw, and used to infect large numbersof permissive cells. The permissive cells can be grown in various tissueculture flasks, for example, 50×150 cm² flasks using various media, suchas Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad,Calif.)) containing 10% fetal bovine serum (Biowhitaker, Walkersvile,Md.) and 10 mM magnesium chloride (Sigma, St Louis, Mo.). The infectedcells can be harvested between 12 and 48 hours after infection or whencomplete cytopathic effects (CPE) are noticed, and are collected bycentrifugation at 1500 rpm for 10 minutes at 4° C. The cell pellet isresuspended in the cell culture supernatant and is subjected to multiplecycles of freeze and thaw. The resulting CVL is clarified bycentrifugation at 1500 rpm for 10 minutes at 4° C. Virus can be purifiedby gradient centrifugation. For example, two rounds of CsCl gradientscan suffice for SVV purification: a one-step gradient (density of CsCl1.24 g/ml and 1.4 g/ml) followed by one continuous gradientcentrifugation (density of CsCl 1.33 g/ml). The purified virusconcentration is determined spectrophotometrically, assuming1A260=9.5×10¹² particles (Scraba D. G., and Palmenberg, A. C. 1999.Cardioviruses (Picornaviridae). In: Encyclopedia of Virology, Secondedition, R. G. Webster and A Granoff, Eds). Infectivity titers ofpurified virus are also determined by a standard plaque and/or tissueculture infective dose 50 (TCID₅₀) assay using PER.C6 or any othersuitable cell type. The yield of SVV from PER.C6 cells are greater than200,000 particles per cell with particles to PFU ratio of about 100. Theyields of SVV from other permissive cells (H446-ATCC#HTB-171) may be atleast this high or higher. SVV can also be purified by columnchromatography.

In addition, several steps in a commercially attractive large scale GoodManufacturing Processes (GMP) are applicable to the purification of SVV.The invention also contemplates methods for purifying SVV that are basedon methods for purifying adenoviruses. These methods include isolatingSVV based on its density, since SVV has a very similar density toadenovirus and can be co-purified with adenovirus.

Methods for Detecting and Studying Tumors:

The present invention provides methods for detecting tumor or neoplasticcells in a patient using the viruses of the present invention. Cellularsamples can be obtained from a patient and screened by incubating thesample with an epitope-tagged SVV (or other tumor-specific virusesprovided by the invention, i.e., tumor-specific mutant cardioviruses),and then screening the sample for bound SVV by detecting the epitopetag. Alternatively, the sample can be screened by detecting whether theSVV causes any cellular lysis. If SVV does cause cellular lysis, or ifSVV can bind specifically to cells in the sample, this would indicatethe possibility that the sample contains neoplastic or tumor cells knownto be capable of being bound and/or infected by SVV.

Additionally, SVV can be used in a method for detecting a tumor cell invivo. In such a method, epitope-tagged SVV can first be inactivated in amanner where SVV can still bind to tumor cells specifically but cannotreplicate. Tumor cells that have bound SVV can be detected by assayingfor the epitope tag. Detection of the epitope tag can be accomplished byantibodies that specifically bind the epitope, where the antibodies areeither labeled (for example, fluorescently) or where the antibodies canthen be detected by labeled secondary antibodies.

The present methods of detection encompass detecting any type of tumoror neoplastic cell that is specifically targeted by any virus of thepresent invention. Specific tumor types include, for example,neuroendocrine-type tumors, such as retinoblastomas, SCLC,neuroblastomas glioblastomas and medulloblastomas.

The present invention also provides the use of SVV as a tool to studytumor cells. SVV selectively destroys some tumor cell types, and hasvery little, if any, toxic effects on non-tumor cells. Because of thesecharacteristics, SVV can be used to study tumors and possibly discover anew tumor specific gene and/or pathway. In other words, there is somecharacteristic of the tumor cells that allows replication of SVV,wherein normal cells do not exhibit said characteristic. Uponidentification of a new tumor specific gene and/or pathway, therapeuticantibodies or small molecules can then be designed or screened todetermine whether these reagents are anti-tumor agents.

The present invention also provides a method for identifying all typesof cancers that respond to SVV. In one embodiment, the method foridentifying SVV-responsive cells comprises obtaining cells, contactingsaid cells with SVV and detecting cell killing or detecting viralreplication. Cell killing can be detected using various methods known toone skilled in the art (e.g., MTS assay, see High-Throughput sectionherein). Methods of detecting virus replication are also known to oneskilled in the art (e.g., observance of CPE, plaque assay, DNAquantification methods, FACS to detect quantity of virus in the tumorcells, RT-PCR assays to detect viral RNA, etc.). In one embodiment, thecells are cancer cells. Examples of cancer cells include, but are notlimited to, established tumor cell lines and tumor cells obtained from amammal. In one embodiment, the mammal is a human. In a furtherembodiment, the cells are cancer cells obtained from a human cancerpatient.

The method for identifying SVV-responsive cancer cells may be used todiscover tumor cell lines or tumor tissues that are permissive for SVVreplication. Also, by determining the characteristics of permissivetumor cells, one may be able to identify characteristics of tumor cellsthat cause the cells to be selectively killed by SVV. The discovery ofthese characteristics could lead to new targets for cancer drugs. Also,the methods for identifying SVV responsive cancer cells could be used asa screen for human cancer patients who would benefit from treatment withSVV.

For example, antibodies against SVV or an SVV-like picornavirus(polyclonal, monoclonal, etc.) can be used in a viral binding assay topre-screen patients prior to SVV or SVV-like picornavirus therapy. Thepre-screening can be conducted generally as follows: (1) cells from apatient are isolated, the cells can be from a tumor biopsy for example,(2) the cells are stained with anti-SVV or anti-SVV-like picornavirusantibodies, (3) a secondary antibody conjugated with a marker (such asfluoroscein or some other detectable dye or fluorophore) that isspecific to the anti-SVV or anti-SVV-like picornavirus antibodies isadded (for example, if the antibodies were raised in a rabbit, then thesecondary antibody would be specific for rabbit immunoglobulins), and(4) detection for the marker is conducted—for example, fluorescencemicroscopy can be conducted where the marker is fluorescein. (Step 3 isoptional if the anti-SVV or anti-SVV-like picornavirus antibodies aredirectly conjugated, i.e., where the antibodies are monoclonal. If theantibodies are polyclonal, indirect immunofluorescence—use of asecondary antibody—is suggested.) If the patient's tumor cells arepermissive for SVV or SVV-like picornavirus infection, then the patientis a candidate for SVV or SVV-like picornavirus therapy. In a virusbinding assay, the patient's tumor cells can be determined to bepermissive for SVV if the cells are positive for antibody staining. Forexample, FIGS. 92B-92C shows immunofluorescent images of cellspermissive for SVV and have been infected with SVV.

In pre-screening patients with a viral binding assay, the cell samplefrom the patient can also be a tissue section of a tissue suspected tocontain tumor cells. The tissue section can then be prepared intosections and incubated with SVV prior to histochemistry with anti-SVV oranti-SVV-like picornavirus antibodies.

The invention also provides methods of detecting SVV. In one embodiment,the detection assay is based on antibodies specific to SVV polypeptideepitopes. In another embodiment, the detection assay is based on thehybridization of nucleic acids. In one embodiment, RNA is isolated fromSVV, labeled (e.g., radioactive, chemiluminsecence, fluorescence, etc.)to make a probe. RNA is then isolated from test material, bound tonitrocellulose (or a similar or functionally equivalent substrate),probed with the labeled SVV RNA, and the amount of bound probe detected.Also, the RNA of the virus may be directly or indirectly sequenced and aPCR assay developed based on the sequences. In one embodiment, the PCRassay is a real time PCR assay.

Methods for Making Viruses with Altered Tropism:

The present invention provides methods for constructing SVV mutants (orvariants or derivatives) where these mutants have an altered cell-typetropism. SVV-like picornaviruses may also be mutated in order to providea particular cell-type tropism. Specifically, SVV and SVV-likepicornavirus mutants are selected for their ability to specifically bindand/or kill tumor or neoplastic cells that are known to be resistant towild-type SVV or wild-type SVV-like picornavirus binding.

The native or wild-type SVV has a simple genome and structure that allowthe modification of the native virus to target other cancer indications.These new derivatives have an expanded tropism toward non-neural cancersand still maintain the high therapeutic index found in the native SVV.One possible means of targeting is the inclusion of tissue-specificpeptides or ligands onto the virus.

To select cancer-targeting viral candidates, the present inventionprovides methods to construct and screen an oncolytic virus library witha genetic insertion that encodes a random peptide sequence in the capsidregion of the native SVV. A random peptide library with a diversity of10⁸ is believed to be sufficient and should yield peptides thatspecifically direct the virus to tumor tissue.

Various studies have shown that tumor cells exhibit differentcharacteristics from normal cells such as: (1) tumor cells have morepermeable cell membranes; (2) tumors have specific stromal cell typessuch as angiogenic endothelial cells which have previously been shown toexpress cell type specific receptors; and (3) tumor cells differentiallyexpress certain receptors, antigens and extracellular matrix proteins(Arap, W. et al., Nat. Med., 2002, 8(2): 121-127; Kolonin, M. et al.,Curr. Opin. Chem. Biol., 2001, 5(3): 308-313; St. Croix, B. et al.,Science, 2000, 289(5482): 1997-1202). These studies demonstrated thattumor and normal tissues are distinct at the molecular level andtargeted drug delivery and treatment of cancer is feasible.Specifically, several peptides selected by homing to blood vessels inmouse models have been used for targeted delivery of cytotoxic drugs(Arap, W. et al., Science, 1998, 279(5349): 377-380), pro-apoptoticpeptides (Ellerby, H. M. et al., Nat. Med., 1999, 17(8): 768-774),metalloprotease inhibitor (Koivunen, E. et al., Nat. Biotechnol, 1999,17(8): 768-774), cytokine (Curnis, F. et al., Nat. Biotechnol., 2000,18(11): 1185-1190), fluorophores (Hong. F. D. and Clayman, G. L., CancerRes., 2000, 60(23): 6551-6556) and genes (Trepel, M. et al., Hum. GeneTher., 2000, 11(14): 1971-1981). The tumor-targeting peptides haveproven to increase the efficacy and lower the toxicity of the parentaldrugs.

A library of SVV derivatives can be generated by the insertion of arandom peptide sequence into the capsid region of the virus. As shown inFIG. 57, a vector is first generated that contains the SVV capsidregion, i.e., “pSVV capsid.” This capsid vector can then be mutagenized,for example, by cutting the vector with a restriction enzyme that cutsDNA at random positions, i.e., CviJI (a blunt cutter). The vector is cutat numerous positions, and DNA that has been cut only once by CviJI canbe isolated by gel-purification (see FIG. 57). This isolated populationof DNA contains a plurality of species that have been cut in the capsidregion at different locations. This population is then incubated witholigonucleotides and ligase, such that a percentage of theoligonucleotides will be ligated into the capsid region of the vector ata number of different positions. In this manner, a library of mutant SVVcapsids can be generated.

The oligonucleotides that are inserted into the capsid encoding regioncan be either random oligonucleotides, non-random oligonucleotides(i.e., the sequence of the oligonucleotide is pre-determined), orsemi-random (i.e., a portion of the oligonucleotide is pre-determinedand a portion has a random sequence). The non-random aspect of thecontemplated oligonucleotides can comprise an epitope-encoding region.Contemplated epitopes include, but are not limited to, c-myc—a 10 aminoacid segment of the human protooncogene myc (EQKLISEEDL (SEQ ID NO: 35);HA—haemoglutinin protein from human influenza hemagglutinin protein(YPYDVPDYA (SEQ ID NO: 36)); and His₆ (SEQ ID NO:116)—a sequenceencoding for six consecutive histidines.

The library of mutant capsid polynucleotides (for example, “pSVV capsidlibrary” in FIG. 57) can then be digested with restriction enzymes suchthat only the mutant capsid encoding region is excised. This mutantcapsid encoding region is then ligated into a vector containing thefull-length genomic sequence minus the capsid encoding region (see FIG.58, for example). This ligation generates a vector having a full-lengthgenomic sequence, where the population (or library) of vectors comprisea plurality of mutant capsids. In FIG. 58, this library of SVV mutantscomprising different capsids is denoted as “pSVVFL capsid.” The pSVVFLcapsid vector library is then linearized and in vitro transcribed inorder to generate mutant SVV RNA (see FIG. 59). The mutant SVV RNA isthen transfected into a permissive cell line such that those SVVsequences that do not possess a debilitating mutation in its capsid aretranslated by the host cells to produce a plurality of mutant SVVparticles. In FIG. 59, the plurality of mutant SVV particles are denotedas a “SVV capsid library.”

The peptide encoded by the oligonucleotide inserted into the capsidencoding region can serve as a targeting moiety for specific viralinfection. The viruses that target a specific type of cancer wouldselectively infect only those cancer cells that have a receptor to thepeptide, replicate in those cells, kill those cells, and spread to onlythose same types of cells. This methodology enables the identificationof novel tumor-targeting peptides and ligands, tumor-selectivereceptors, therapeutic SVV derivatives and other virus derivatives,including picornavirus derivatives.

In vitro and in vivo screening of SVV mutant libraries have severaladvantages over other technologies such as peptide bead libraries andphage display. Unlike these other technologies, the desirable candidatehere, i.e. an SVV derivative that selectively binds to a cancer cell,will replicate in situ. This replication-based library approach hasnumerous advantages over prior methods of discovering new cell bindingmoieties, such as phage display. First, the screening of a SVV libraryis based on replication. Only the desired viral derivatives canreplicate in the target tissue, in this case certain cancer cells. Thescreening/selection process will yield very specific viral candidatesthat have both the targeting peptide moiety and may be a cancertherapeutic itself. On the contrary, phage display screens will onlyresult in binding events and yields only the targeting peptidecandidates. Thus, SVV library screening offers a much faster andselective approach. Second, during in vitro or in vivo phage displayscreening, phages recovered from the target cells have to be amplifiedin bacteria, while SVV derivatives can be directly recovered andpurified from infected cells (or from the culture supernatant oflytically infected cells). Third, SVV has a smaller genome that renderseasier manipulability; thus it is possible to randomly insert thegenetic information into the capsid region to ensure an optimizedinsertion. Therefore, construction and screening of the SVV library hasa high possibility to produce highly effective viral derivatives. Thesederivatives are designed and screened to specifically infect cancerswith non-neural properties.

The insertion of oligonucleotides into the capsid encoding region willresult in the generation of some defective mutants. Mutants may bedefective in the sense that the insertion of an oligonucleotide sequencecan result in a stop codon, such that the viral polyprotein will not beproduced. Also, mutants may be defective in the sense that the insertionof an oligonucleotide sequence may result in the alteration of thecapsid structure such that capsid can no longer be assembled. Todecrease the probability that the insertion of oligonucleotide sequencesmay result in stop codon or untenable capsid structure, randomoligonucleotides can be designed such that they do not encode for stopcodons or for certain amino acids using methods such as TRIM.

To determine whether there is an optimal insertion point in the capsidregion for oligonucleotides, one can generate an RGD-SVV library (seeExample 16). The polynucleotide encoding the SVV capsid is randomly cut,for example, with CviJI. The randomly linearized capsid polynucleotidesare then ligated to oligonucleotides encoding at least the RGD aminoacid sequence (arginine-glycine-aspartic acid). These RGD-capsidsequences are then ligated into SVV full-length sequence vectors thatare missing the capsid sequence. RGD-SVV derivatives viruses areproduced and tested for their ability to infect and replicate in certainintegrin-expressing cell lines (as the RGD peptide has been shown totarget entities to integrin receptors). The RGD-SVV derivatives that aresuccessful in infecting the integrin-expressing cell lines are thenanalyzed to determine whether there is a predominant insertion site forthe RGD oligonucleotide. This site can then be used for site-directedinsertion of random, non-random or semi-random oligonucleotides.

Further, in comparing portions of the capsid encoding region between SVVand other picornaviruses (see FIG. 28), there are various non-boxedregions between the viruses where the sequence similarity is at itslowest. These regions may be important in contributing to the differenttropisms between the viruses. Thus, these regions may be candidatelocations for oligonucleotide insertion mutagenesis of the SVV capsid(and for other viral capsids).

Inactivated SVV as a Tumor-Specific Therapeutic:

Since SVV and SVV-capsid derivatives can target specific tumorcell-types and/or tissues, the SVV particle itself can be used as adelivery vehicle for therapeutics. In such a method, the need for theoncolytic abilities of SVV becomes optional, as the deliveredtherapeutic can kill the targeted tumor cell.

For example, the wild-type SVV can be inactivated such that the virus nolonger lyses infected cells, but where the virus can still specificallybind and enter targeted tumor cell-types. There are many standardmethods known in the art to inactivate the replicative functions ofviruses. For example, whole virus vaccines are inactivated by formalinor β-propiolactone such that the viruses cannot replicate. The wild-typeSVV may itself contain peptides that cause the apoptosis of cells.Alternatively, SVV can be irradiated. However, irradiated viruses shouldfirst be tested to ensure that they are still capable of specificallytargeting tumor cells, as certain irradiation conditions may causeprotein, and thus capsid, alterations. Further, mutant SVVs can begenerated where the packaging signal sequence is deleted. These SVVmutants are able to specifically bind and enter target cells, butreplicated SVV genomic RNA will not be packaged and assembled intocapsids. However, this method may prove to be useful as initial entry ofthese mutant SVVs will cause host-protein synthesis shut-off such thattumor-cell death is still achieved.

Derivative SVVs having mutant capsids can also be inactivated and usedto kill cancer cells. Derivative SVVs with oligonucleotides encodingepitope tags inserted into the capsid region can be used as vehicles todeliver toxins to tumor cells. As described herein, derivative SVVs canbe randomly mutagenized and screened for tumor-specific tropisms. Toxinscan be attached to the epitope tags, such that the virus delivers thetoxin to tumor cells. Alternatively, therapeutic antibodies thatspecifically bind to the epitope tag can be used, such that the virusdelivers the therapeutic antibody to the tumor cell.

High-Throughput Screening:

The present invention encompasses high-throughput methods for screeningviruses that have the ability to specifically infect differentcell-lines. The specificity of infection can be detected by assaying forcytopathic effects. For example, a number of different tumor cell-linescan be grown in different wells of a multi-well plate that is amenablefor high-throughput screening, for example a 384 well-plate. To eachwell, a sample of virus is added to test whether the cells are killed byvirus-mediated lysis. From those wells that show cytopathic effects, themedia is collected such that any viruses in the media can be amplifiedby infecting permissive cell lines in flasks or large tissue cultureplates. The viruses are grown such that the RNA can be isolated and thesequence analyzed to determine sequence mutations that may beresponsible for providing a tumor cell-type specific tropism for avirus.

Various colorimetric and fluorometric methods can quickly assaycytopathic effects, including fluorescent-dye based assays, ATP-basedassays, MTS assays and LDH assays. Fluorescent-dye based assays caninclude nucleic acid stains to detect dead-cell populations, ascell-impermeant nucleic acid stains can specifically detect dead-cellpopulations. If it is desired to simultaneously detect both live-celland dead-cell populations, nucleic acid stains can be used incombination with intracellular esterase substrates, membrane-permeantnucleic acid stains, membrane potential-sensitive probes, organelleprobes or other cell-permeant indicators to detect the live-cellpopulation. For example, Invitrogen (Carlsbad, Calif.) offers variousSYTOX™ nucleic acid stains that only penetrate cells with compromisedplasma membranes. Ethidium bromide and propidium iodide can also be usedto detect dead or dying cells. These stains are high-affinity nucleicacid stains that can be detected by any light-absorbance reader

For example, lysis can be based on the measurement of lactatedehydrogenase (LDH) activity released from the cytosol of damaged cellsinto the supernatant. To detect the presence of LDH in cell culturesupernatants, a substrate mixture can be added such that LDH will reducethe tetrazolium salt INT to formazan by a coupled enzymatic reaction.The formazan dye can then be detected by a light-absorbance reader.Alternatively, an MTS assay[3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt] using phenzine methosulfate (PMS) as the electron couplingreagent can also be used to detect cytotoxicity. Promega (Madison, Wis.)offers a CellTiter 96® AQ_(ueous) One Solution Cell Proliferation Assaykit where the solution reagent is added directly to culture wells,incubated for 1-4 hours and then absorbance is recorded at 490 nm. Thequantity of formazan product as measured by the amount of 490 nmabsorbance is directly proportional to the number of living cells inculture.

There are numerous high-throughput devices for reading light-absorbance.For example, SpectraMax Plus 384 Absorbance Platereader (MolecularDevices) can detect wavelengths from 190-1000 nm in 1 nm increments. Thedevice can read 96-well microplates in 5 seconds and 384-wellmicroplates in 16 seconds for ultra fast sample throughput.

Virus replication can also be assayed as an indication of successfulinfection, and such detection methods can be used in a high-throughputmanner. For example, real-time RT-PCR methods can be used to detect thepresence of virus transcripts in cell-culture supernatants. Uponreverse-transcription of viral RNA into cDNA, the cDNA can be amplifiedand detected by PCR with the use of double-stranded DNA-binding dyes(for example, SYBR® Green, Qiagen GmbH, Germany). The amount of PCRproduct can then be directly measured using a fluorimeter.

Viruses from the wells showing cytopathic effects are grown up andtested in further in vitro (re-testing of tumor and normal cell lines)and in vivo models (testing whether the virus can kill explanted tumorsin mice).

Antibodies:

The present invention is also directed to antibodies that specificallybind to the viruses of the present invention, including the proteins ofthe viruses. Antibodies of the present invention include naturallyoccurring as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric antibodies, bifunctionalantibodies and humanized antibodies, as well as antigen-bindingfragments thereof. Such non-naturally occurring antibodies can beconstructed using solid phase peptide synthesis, can be producedrecombinantly or can be obtained, for example, by screeningcombinatorial libraries consisting of variable heavy chains and variablelight chains (Huse et al., Science 246:1275-1281, 1989). These and othermethods of making, for example, chimeric, humanized, CDR-grafted, singlechain, and bifunctional antibodies are well known to those skilled inthe art (Winter and Harris, Immunol. Today 14:243-246, 1993; Ward etal., Nature 341:544-546, 1989; Harlow and Lane, Antibodies: A laboratorymanual, Cold Spring Harbor Laboratory Press, 1988); Hilyard et al.,Protein Engineering: A practical approach, IRL Press 1992; Borrabeck,Antibody Engineering, 2d ed., Oxford University Press 1995). Antibodiesof the invention include intact molecules as well as fragments thereof,such as Fab, F(ab′)2, and Fv which are capable of binding to an epitopicdeterminant present in a polypeptide of the present invention.

Where a peptide portion of a SVV polypeptide of the invention (i.e., anypeptide fragment from SEQ ID NO:2 or SEQ ID NO:169) or peptide portionof another viral polypeptide of the invention used as an immunogen forantibody generation is non-immunogenic, it can be made immunogenic bycoupling the hapten to a carrier molecule such as bovine serum albumin(BSA) or keyhole limpet hemocyanin (KLH), or by expressing the peptideportion as a fusion protein. Various other carrier molecules and methodsfor coupling a hapten to a carrier molecule are well known in the art(for example, by Harlow and Lane, supra, 1988). Methods for raisingpolyclonal antibodies, for example, in a rabbit, goat, mouse or othermammal, are well known in the art (see, for example, Green et al.,“Production of Polyclonal Antisera,” in Immunochemical Protocols,Manson, ed., Humana Press 1992, pages 1-5; Coligan et al., “Productionof Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters,” in Curr.Protocols Immunol. (1992), section 2.4.1).

Monoclonal antibodies also can be obtained using methods that are wellknown and routine in the art (Kohler and Milstein, Nature 256:495, 1975;Coligan et al., supra, 1992, sections 2.5.1-2.6.7; Harlow and Lane,supra, 1988). For example, spleen cells from a mouse immunized with avirus, viral polypeptide or fragment thereof, can be fused to anappropriate myeloma cell line to produce hybridoma cells. Clonedhybridoma cell lines can be screened using, for example, labeled SVVpolypeptide to identify clones that secrete monoclonal antibodies havingthe appropriate specificity, and hybridomas expressing antibodies havinga desirable specificity and affinity can be isolated and utilized as acontinuous source of the antibodies. Polyclonal antibodies similarly canbe isolated, for example, from serum of an immunized animal. Suchantibodies, in addition to being useful for performing a method of theinvention, also are useful, for example, for preparing standardizedkits. A recombinant phage that expresses, for example, a single chainantibody also provides an antibody that can be used for preparingstandardized kits. Monoclonal antibodies, for example, can be isolatedand purified from hybridoma cultures by a variety of well establishedtechniques, including, for example, affinity chromatography withProtein-A SEPHAROSE gel, size exclusion chromatography, and ion exchangechromatography (Barnes et al., in Meth. Mol. Biol. 10:79-104, HumanaPress 1992); Coligan et al., supra, 1992, see sections 2.7.1-2.7.12 andsections 2.9.1-2.9.3).

An antigen-binding fragment of an antibody can be prepared byproteolytic hydrolysis of a particular antibody, or by expression of DNAencoding the fragment. Antibody fragments can be obtained by pepsin orpapain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)2. Thisfragment can be further cleaved using a thiol-reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly (see, forexample, Goldenberg, U.S. Pat. No. 4,036,945 and U.S. Pat. No.4,331,647; Nisonhoff et al., Arch. Biochem. Biophys. 89:230. 1960;Porter, Biochem. J. 73:119, 1959; Edelman et al., Meth. Enzymol., 1:422(Academic Press 1967); Coligan et al., supra, 1992, see sections2.8.1-2.8.10 and 2.10.1-2.10.4).

Another example of an antigen binding fragment of an antibody is apeptide coding for a single complementarity determining region (CDR).CDR peptides can be obtained by constructing polynucleotides encodingthe CDR of an antibody of interest. Such polynucleotides can beprepared, for example, using the polymerase chain reaction to synthesizea variable region encoded by RNA obtained from antibody-producing cells(for example, Larrick et al., Methods: A Companion to Methods inEnzymology 2:106, 1991).

The antibodies of the invention are suited for use, for example, inimmunoassays in which they can be utilized in liquid phase or bound to asolid phase carrier. In addition, the antibodies in these immunoassayscan be detectably labeled in various ways. Examples of types ofimmunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

There are many different labels and methods of labeling antibodies knownto those of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, fluorescent compounds, colloidal metals, chemiluminescentcompounds, phosphorescent compounds, and bioluminescent compounds. Thoseof ordinary skill in the art will know of other suitable labels forbinding to the antibody, or alternatively to the antigen, or will beable to ascertain such, using routine experimentation.

As various changes can be made in the above methods and compositionswithout departing from the scope and spirit of the invention asdescribed, it is intended that all subject matter contained in the abovedescription, shown in the accompanying drawings, or defined in theappended claims be interpreted as illustrative, and not in a limitingsense.

EXAMPLES

The examples described below are provided to illustrate the presentinvention and are not included for the purpose of limiting theinvention.

Example 1 Amplification and Purification of Virus

Cultivation of SVV in PER.C6 cells: SVV is plaque purified once and awell isolated plaque is picked and amplified in PER.C6 cells (Fallaux etal., 1998). A crude virus lysate (CVL) from SVV infected PER.C6 cells ismade by three cycles of freeze and thaw and used to infect PER.C6 cells.PER.C6 cells are grown in 50×150 cm² T.C. flasks using Dulbecco'smodified Eagle medium (DMEM, Invitrogen, Carlsbad, Calif., USA))containing 10% fetal bovine serum (Biowhitaker, Walkersvile, Md., USA)and 10 mM magnesium chloride (Sigma, St Louis, Mo., USA). The infectedcells harvested 30 hr after infection when complete CPE is noticed andare collected by centrifugation at 1500 rpm for 10 minutes at 4° C. Thecell pellet is resuspended in the cell culture supernatant (30 ml) andis subjected to three cycles of freeze and thaw. The resulting CVL isclarified by centrifugation at 1500 rpm for 10 minutes at 4° C. Virus ispurified by two rounds of CsCl gradients: a one-step gradient (densityof CsCl 1.24 g/ml and 1.4 g/ml) followed by one continuous gradientcentrifugation (density of CsCl 1.33 g/ml). The purified virusconcentration is determined spectrophotometrically, assuming1A₂₆₀=9.5×10¹² particles (Scraba D. G., and Palmenberg, A. C. 1999.Cardioviruses (Picornaviridae). In: Encyclopedia of Virology, Secondedition, R. G. Webster and A Granoff, Eds). Titers of purified virus arealso determined by a standard plaque assay using PER.C6 cells. The yieldof SVV from PER.C6 cells are greater than 200, 000 particles per cellwith particles to PFU ratio of about 100. The yields of SVV from otherpermissive cells (H446-ATCC# HTB-171) may be at least this high orhigher.

Example 2 Electron Microscopy

SVV is mounted onto formvar carbon-coated grids using the directapplication method, stained with uranyl acetate, and examined in atransmission electron microscope. Representative micrographs of thevirus are taken at high magnification. For the transmission electronmicroscope, ultra-thin sections of SVV-infected PER.C6 cells are cutfrom the embedded blocks, and the resulting sections are examined in thetransmission electron microscope.

The purified SVV particles are spherical and about 27 nm in diameter,appearing singly or in small aggregates on the grid. A representativepicture of SVV is shown in FIG. 2. In some places, broken viralparticles and empty capsids with stain penetration are also seen.Ultrastructural studies of infected PER.C6 cells revealed crystallineinclusions in the cytoplasm. A representative picture of PER.C6 cellsinfected with SVV is shown in FIG. 3. The virus infected cells revealeda few large vesicular bodies (empty vesicles).

Example 3 Nucleic Acid Isolation of SVV

RNA Isolation: SVV genomic RNA was extracted using guanidium thiocyanateand a phenol extraction method using Trizol (Invitrogen). Isolation wasperformed according to the supplier's recommendations. Briefly, 250 μlof the purified SVV was mixed with 3 volumes TRIZOL and 240 μl ofchloroform. The aqueous phase containing RNA was precipitated with 600μl isopropanol. The RNA pellet was washed twice with 70% ethanol, driedand dissolved in DEPC-treated water. The quantity of RNA extracted wasestimated by optical density measurements at 260 nm. An aliquot of RNAwas resolved through a 1.25% denaturing agarose gel (Cambrex BioSciences Rockland Inc., Rockland, Me. USA) and the band was visualizedby ethidium bromide staining and photographed (FIG. 4).

cDNA synthesis: cDNA of the SVV genome was synthesized by RT-PCR.Synthesis of cDNA was performed under standard conditions using 1 μg ofRNA, AMV reverse transcriptase, and random 14-mer oligonucleotide oroligo-dT. Fragments of the cDNA were amplified, cloned into plasmids andthe clones are sequenced.

Example 4 SVV Sequence Analysis and Epidemiology

Part I: SVV SEQ ID NO:1

The nucleotide sequence of SVV SEQ ID NO:1 was analyzed to determine itsevolutionary relationship to other viruses. The translated product (SEQID NO:2) for this ORF was picornavirus-like and reached from the middleof VP2 to the termination codon at the end of the 3D polymerase and was1890 amino acids in length (FIG. 5A-5E and 7A-7B). The 3′ untranslatedregion (UTR), nucleotides 5671-5734, which follows the ORF is 64nucleotides (nt) in length, including the termination codon andexcluding the poly(A) tail of which 18 residues are provided (FIG. 5E).

Preliminary comparisons (not shown) of three partial genome segments ofSVV had revealed that SVV was most closely related members of the genusCardiovirus (family Picornaviridae). Therefore an alignment of thepolyprotein sequences of SVV, encephalomyocarditis virus (EMCV; speciesEncephalomyocarditis virus, Theiler's murine encephalomyelitis virus(TMEV; species Theilovirus), Vilyuisk human encephalomyelitis virus(VHEV; species Theilovirus) and a rat TMEV-like agent (TLV; speciesTheilovirus) was constructed (FIG. 28). From this alignment, the SVVpolyprotein processing was compared to the polyprotein processing of themost closely related members of the Cardiovirus genus. Cleavage sitesbetween the individual polypeptides is demarcated by the “I” characterin FIG. 28.

In picornaviruses, most polyprotein cleavages are carried out by one ormore virus-encoded proteases, although in cardio-, aphtho-, erbo- andteschoviruses the cleavage between P1-2A and 2B is carried out by apoorly understood cis-acting mechanism related to the 2A sequence itselfand critically involving the sequence “NPG/P”, where “I” represents thebreak between the 2A and 2B polypeptides (Donnelly et al., 1997, J. Gen.Virol. 78: 13-21). One of the parechoviruses, Ljungan virus, has thissequence (NPGP) present upstream of a typical parechovirus 2A and iseither an additional 2A or is the C-terminal end of the P1 capsidregion. In all nine currently recognised picornavirus genera, 3C^(pro)carries out all but the cis-acting self-cleaving reactions (i.e. 2Acleaves at its N-terminus in entero- and rhinoviruses and L cleaves atits C-terminus inaphthoviruses and erboviruses). The post-assemblycleavage of the capsid polypeptide VP0 to VP4 and VP2 is not carried outby 3C^(pro), but by an unknown mechanism which may involve the virusRNA. The VP0 cleavage does not occur in parechoviruses and kobuviruses.The normal cardiovirus 3C^(pro) cleavage site has either a glutamine (Q)or glutamate (E) at the −1 position and glycine (G), serine (S), adenine(A) or asparagine (N) at the +1 position (Table 2). The cleavages of theSVV polyprotein conform to this pattern except for the VP3/VP1 sitewhich is histidine (H)/serine (S) (Table 2); however, H/S is probablypresent as the cleavage site between 3A and 3B^(VPg) in at least onestrain of equine rhinitis A virus (ERAV; genus Aphthovirus) (Wutz etal., 1996, J. Gen. Virol. 77:1719-1730).

TABLE 2 Cleavage sites of SVV and cardioviruses Between SVV EMCV TMEVRat TLV VHEV L VP4 Not  LQ/GN PQ/GN PQ/GN PQ/GN known (SEQ (SEQ (SEQ (SEQ  ID NO: ID NO: ID NO: ID NO: 125) 138) 152) 163) VP4  VP2 Not LA/DQLL/DQ LL/DQ LL/DE      known (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO:ID NO: 126) 139) 153) 164)   LM/DQ (SEQ ID NO: 140) VP2  VP3 EQ/GP RQ/SPAQ/SP PQ/SP PQ/SP       (SEQ (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO:ID NO: ID NO: 117) 127) 141) 154) 165) VP3  VP1 FH/ST PQ/GV PQ/GV PQ/GVPQ/GV      (SEQ (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: ID NO:118) 128) 142) 155) 166) PQ/GI   (SEQ ID NO: 143) PQ/GS (SEQ   ID NO:144) VP1  2A KQ/KM LE/SP LE/NP LQ/NP LE/NP       (SEQ (SEQ (SEQ (SEQ(SEQ ID NO: ID NO: ID NO: ID NO: ID NO: 119) 129) 145) 156) 167) 2A 2BNPG/P* NPG/P* NPG/P* NPG/P* Nk      (SEQ (SEQ (SEQ (SEQ ID NO: ID NO:ID NO: ID NO: 111) 130) 146) 157) 2B 2C MQ/GP QQ/SP PQ/GP AQ/SP Nk     (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 120) 131) 147) 158) 2C3A LQ/SP AQ/GP AQ/SP AQ/SP Nk      (SEQ (SEQ (SEQ (SEQ ID NO: ID NO:ID NO: ID NO: 121) 132) 148) 159) AQ/AP   (SEQ ID NO: 133) 3A 3B SE/NAEQ/GP EQ/AA EQ/AA Nk      (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO:ID NO: 122) 134) 149) 160) 3B 3C MQ/QP IQ/GP IQ/GG IQ/GG Nk      (SEQ(SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 123) 135) 150) 161)   VQ/GP(SEQ ID NO: 136) 3C 3D MQ/GL PQ/GA PQ/GA PQ/GA Nk     (SEQ (SEQ (SEQ(SEQ ID NO:  ID NO: ID NO: ID NO: 124) 137) 151) 162) *,the breakbetween 2A and 2B is not a cleavage event

Primary cleavages (P1/P2 and P2/P3) of SVV: These primary cleavageevents are predicted to occur in a similar fashion to cardio-, aphtho-,erbo- and teschoviruses, involving separation of P1-2A from 2B by anovel mechanism involving the sequence NPG/P (SEQ ID NO:111) and atraditional cleavage event by 3C^(pro) between 2BC and P3 (Table 2).

P1 cleavages: Cleavages within the SVV P1 capsid coding region wererelatively easy to predict by alignment with sequence with EMCV and TMEV(Table 2).

P2 cleavages: The 2C protein is involved in RNA synthesis. The 2Cpolypeptide of SVV contains NTP-binding motifs GxxGxGKS/T (SEQ IDNO:112) (domain A) and hyhyhyxxD (in which by is any hydrophobicresidue; domain B) present in putative helicases and all picornavirus2Cs (FIG. 29).

P3 cleavages: Prediction of the P3 cleavage sites was also relativelystraightforward. Little is known about the function of the 3Apolypeptide. However, all picornavirus 3A proteins contain a putativetransmembrane alpha-helix. Primary sequence identity is low in thisprotein between SVV and cardioviruses (See FIG. 28 between positions1612 to 1701).

The genome-linked polypeptide, VPg, which is encoded by the 3B region,shares few amino acids in common with the other cardioviruses, however,the third residue is a tyrosine, consistent with its linkage to the 5′end of the virus genome (Rothberg et al., 1978). See FIG. 28 betweenpositions 1703 and 1724.

The three-dimensional structure of four picornavirus 3C cysteineproteases have been solved and the active-site residues identified (HAV,Allaire et al., 1994, Nature, 369: 72-76; Bergmann et al., 1997, J.Virol., 71: 2436-2448; PV-1, Mosimann et al., 1997, J. Mol. Biol., 273:1032-1047; HRV-14, Matthews et al., 1994, Cell, 77: 761-771; and HRV-2,Matthews et al., 1999, Proc. Natl. Acad. Sci. USA, 96: 11000-11007). Thecysteine bolded in FIG. 29 is the nucleophile, while the first boldedhistidine is the general base and the specificity for glutamine residuesis defined mainly by the second bolded histidine; all three residues areconserved in the SVV sequence (FIG. 29) and all other knownpicornaviruses (FIG. 28; for 3C sequence comparison see betweenpositions 1726 and 1946).

The 3D polypeptide is the major component of the RNA-dependent RNApolymerase and SVV contains motifs conserved in picorna-like virusRNA-dependent RNA polymerases, i.e. KDEL/IR (SEQ ID NO:113), PSG, YGDD(SEQ ID NO:114) and FLKR (SEQ ID NO:115) (FIG. 3; FIG. 28 betweenpositions 1948 and 2410).

Myristoylation of the N-terminus of P1: In most picornaviruses the P1precursor polypeptide is covalently bound by its N-terminal glycineresidue (when present the N-terminal methionine is removed) to amolecule of myristic acid via an amide linkage (Chow et al., 1987,Nature, 327: 482-486). Consequently the cleavage products VP0 and VP4which contain the P1 N-terminus are also myristoylated. Thismyristoylation is carried out by myristoyl transferase which recognisesan eight amino acid signal beginning with glycine. In picornaviruses, afive residue consensus sequence motif, G-x-x-x-T/S, has been identified(Palmenberg, 1989, In Molecular Aspects of Picornavirus Infection andDetection, pp. 211-241, Ed. Semler & Ehrenfeld, Washington D.C., Amer.Soc. for Micro.). Parechoviruses (Human parechovirus and Ljungan virus)as well as not having a maturation cleavage of VP0 are apparently notmyristoylated, however, there appears to be some type of moleculeblocking the N-terminus of VP0 for these viruses.

Comparisons of the Individual SVV Polypeptides with the Public SequenceDatabases

Each of the SVV polypeptides (SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18,20 and 22) were compared to the public protein sequence databases usingthe FASTA online program at the European Bioinformatics Institute (EBI;http://www.ebi.ac.uk/). The results (best matches) of these comparisonsare shown in Table 3. The capsid polypeptides (VP2, VP3 and VP1) takenas a whole, along with 2C, 3C^(pro) and 3D^(pol) are most closelyrelated to members of the cardiovirus genus, however, the shortpredicted 2A sequence is closer to that of Ljungan virus (genusParechovirus). A more detailed comparison of the SVV 2A nucleotidesequence with similar sequences is shown in FIG. 28 (see also FIG. 70for 2A-like NPG/P protein comparison).

TABLE 3 Database matches of individual predicted polypeptides of SenecaValley virus SVV Length % % identity Matched polypeptide (aa) identityungapped aa overlap Organism protein L (Leader) No data — — — — — VP4(1A) No data — — — — — VP2 (1B) >142 42.857 44.037 112 TMEV WW VP2 ~51 —~80 EMCV BEL-2887A/91 VP2 VP3 (1C) 239 44.068 46.637 236 EMCV ATCCVR-129B VP3 VP1 (1D) 259 31.086 36.404 267 EMCV M100/1/02 VP1 2A 1471.429 71.429 14 Ljungan virus 174F 2A1 2B 128 39.286 41.509 56Ureaplasma urealyticum Multiple banded antigen 2C 322 38.602 40.190 329EMCV PV21 2C 3A 90 37.838 41.791 74 Chlorobium tepidum TLS* Enolase 2†3B^(VPg) 22 No — — — — matches 3C^(pro) 211 37.089 38.537 213 EMCV-R 3Cprotease 3D^(pol) 462 58.009 58.515 462 EMCV-PV21 3D polymerase *aphotosynthetic, anaerobic, green-sulfur bacterium †2-phosphoglyceratedehydratase 2) (2-phospho-D-glycerate hydro-lyase 2

The significance of the matches of SVV 2B with Ureaplasma urealyticummultiple banded antigen or 3A with Chlorobium tepidum endolase 2 is notclear, however, these relationships maybe worthy of furtherinvestigation.

Phylogenetic Comparison of SVV Polypeptides with Other Picornaviruses

Those SVV polypeptides which could be aligned with the cardioviruses(VP2, VP3, VP1, 2C, 3C and 3D) were compared with the same proteins ofrepresentative members of each of the picornavirus species (Table 4).The programs BioEdit v5.0.9 (Hall, 1999, Nucl. Acids. Symp. Ser., 41:95-98) and Clustal X v1.83 (Thompson et al., 1997, Nucl. Acids Res.,25:4876-4882) were used to make the alignments and to construct distancematrices and unrooted Neighbor-joining trees according to the algorithmof Saitou and Nei (Satiou and Nei, 1987, Mol. Biol. Evol., 4: 406-425).Confidence limits on branches were accessed by bootstrap resampling(1000 pseudo-replicates). The trees were drawn using TreeView 1.6.6(Page, 1996) (FIGS. 31 to 37). The distance matrices used to constructthe trees used values corrected for multiple substitutions, while FIGS.38-44 show the actual percentage amino acid identities. Table 4 showsthe current classification of the family Picornaviridae and therepresentative virus sequences used in these comparisons.

TABLE 4 The taxonomic classification of the picornaviruses used in thecomparisons with SVV. Genus Species Representative virus Abbrev. Acc.No. Enterovirus Poliovirus Poliovirus 1 PV-1 V01149 Human enterovirus ACoxsackievirus A16 CV-A16 U05876 Human enterovirus B Coxsackievirus B5CV-B5 X67706 Human enterovirus C Coxsackievirus A21 CV-A21 D00538 Humanenterovirus D Enterovirus 70 EV-70 D00820 Simian enterovirus A Simianenterovirus A1 SEV-A AF201894 Bovine enterovirus Bovine enterovirus 1BEV-1 D00214 Porcine enterovirus B Porcine enterovirus 9 PEV-9 AF363453New genus? Not yet designated Simian virus 2* SV2 AY064708 Porcineenterovirus A Porcine enterovirus 8* PEV-8 AF406813 Rhinovirus Humanrhinovirus A Human rhinovirus 2 HRV-2 X02316 Human rhinovirus B Humanrhinovirus 14 HRV-14 K02121 Cardiovirus Encephalomyocarditis virusEncephalomyocarditis virus EMCV M81861 Theilovirus Theiler's murineencephalomyelitis TMEV M20562 virus Aphthovirus Foot-and-mouth diseasevirus Foot-and-mouth disease virus O FMDV-O X00871 Equine rhinitis Avirus Equine rhinitis A virus ERAV X96870 Hepatovirus Hepatitis A virusHepatitis A virus HAV M14707 Avian encephalomyelitis-like Avianencephalomyelitis virus AEV AJ225173 viruses Parechovirus Humanparechovirus Human parechovirus 1 HPeV-1 L02971 Ljungan virus Ljunganvirus LV AF327920 Kobuvirus Aichi virus Aichi virus AiV AB040749 Bovinekobuvirus Bovine kobuvirus BKV AB084788 Erbovirus Equine rhinitis Bvirus Equine rhinitis B virus 1 ERBV-1 X96871 Teschovirus Porcineteschovirus Porcine teschovirus 1 PTV-1 AJ011380 *the current taxonomicstatus of SV2 and PEV-8 places them in the enterovirus genus, however,it has been suggested that they may be reclassified in a new genus(Krumbholz et al., 2002; Oberste et al., 2003).

The trees of the individual capsid proteins (FIGS. 31 to 33) are not allrepresentative of the tree produced when the data from all treepolypeptides is combined (FIG. 34). This is probably the result ofdifficulties in aligning the capsid polypeptides, particularly when theyare not full length as is the case for VP2 (FIG. 31). However, the P1,2C, 3C^(pro) and 3D^(pol) trees are all in agreement and show that SVVclusters with EMCV and TMEV.

Seneca Valley Virus as a Member of the Cardiovirus Genus

Clearly the 3D^(pol) of SVV is related to the cardioviruses, almost asclosely as EMCV and TMEV are to each other (FIG. 37; FIG. 44). In theother polypeptides which are generally considered as being relativelyconserved in picornaviruses, 2C and 3C, SVV is also most closely relatedto the cardioviruses although it is not as closely related to EMCV andTMEV as they are to each other (FIG. 42 and FIG. 43, respectively). Inthe outer capsid proteins (taken as a whole), SVV is also most closelyrelated to the cardioviruses and has approximately the same relationshipas the two aphthovirus species, Foot-and-mouth disease virus and Equinerhinitis A virus (˜33%). SVV diverges greatly from the cardioviruses inthe 2B and 3A polypeptides and has no detectable relationship with anyknown picornavirus. However, this is not without precedent; avianencephalomyelitis virus differs considerably from hepatitis A virus(HAV) in 2A, 2B and 3A (Marvil et al., 1999, J. Gen. Virol., 80:653-662)but is tentatively classified within the genus Hepatovirus along withHAV.

Seneca Valley virus is clearly not a typical cardiovirus if EMCV andTMEV are taken as the standard. However, even these two viruses havetheir differences, notably in the 5′ UTR (Pevear et al., 1987, J. Gen.Virol., 61: 1507-1516). However, phylogenetically SVV clusters with EMCVand TMEV in much of its polyprotein (P1, 2C, 3C^(pro) and 3D^(pol)regions). Ultimately, the taxonomic position of SVV within thePicornaviridae will be decided by the Executive Committee (EC) of theInternational Committee for the Taxonomy of Viruses (ICTV) followingrecommendations by the Picornaviridae Study Group and supportingpublished material. There are two options: i) include SVV as a newspecies in the cardiovirus genus; or ii) assign SVV to a new genus.

Part II: SVV SEQ ID NO:168

The full-length genome of SVV (FIGS. 83A-83H; SEQ ID NO:168; Example 15)allowed further epidemiological studies. The results of the furtherepidemiological studies are shown in FIG. 86, where SVV is shown to begenetically related to cardioviruses such as EMCV and TMEV, but in aseparate tree.

The features of the SVV full-length genome with respect to itsuntranslated and coding regions are listed at Table A supra. Thefeatures of the full-length SVV in comparison to EMCV and TMEV-GDVII arelisted in the table below.

EMCV EMCV TMEV-GDVII TMEV-GDVII SVV SVV [M81861] [M81861] [M20562][M20562] Feature nt length aa length nt length aa length nt length aalength 5 ‘UTR 666 — 833 — 1068 — Leader 237 79 201 67 228 76 VP4 213 71210 70 213 71 VP2 852 284 768 256 801 267 VP3 717 239 693 231 696 232VP1 792 264 831 277 828 276 2A 27 9 429 143 426 142 2B 384 128 450 150381 127 2C 966 399 975 325 978 326 3A 270 90 264 88 264 88 3B 66 22 6020 60 20 3D 1386 462 1380 460 1383 461 3’ UTR 71 — 126 — 128 —

The cleavage sites of SVV (based on full-length sequence, see alsobolded amino acids between at protein boundaries in FIGS. 83A-83H) arecompared to the cleavage sites of other cardioviruses in the tablebelow.

Between SVV EMCV TMEV Rat TLV VHEV L VP4 LQ/GN   LQ/GN PQ/GN PQ/GNPQ/GN  (SEQ ID  (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 192) NO: 192)NO: l93) NO: 193) NO: 193) VP4 VP2 LK/DH LA/DQ LL/DQ  LL/DQ LL/DE(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 194) NO: 195) NO: 196)NO: 196) NO: 198) LM/DQ  (SEQ ID  NO: 197) VP2 VP3 EQ/GP RQ/SP AQ/SPPQ/SP PQ/SP (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 117) NO: 199)NO: 200) NO: 201) NO :201) VP3 VP1 FH/ST PQ/GV PQ/GV  PQ/GV PQ/GV(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 118) NO: 202) NO: 202)NO: 202) NO: 202) PQ/GI (SEQ ID NO: 203) PQ/GS (SEQ ID NO: 204) VP1 2AMQ/SG LE/SP LE/NP LQ/NP LE/NP (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 205) NO: 206) NO: 207) NO: 208) NO: 207) 2A 2B NPG/P* NPG/P* NPG/P*NPG/P* unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 111) NO: 111)NO: 111) NO: 111) 2B 2C MQ/GP QQ/SP PQ/GP AQ/SP unknown (SEQ ID (SEQ ID  (SEQ ID  (SEQ ID NO: 120) NO: 209) NO: 210) NO: 200) 2C 3ALQ/SP AQ/GP AQ/SP AQ/SP unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 121)NO: 211) NO: 200) NO: 200) AQ/AP   (SEQ ID  NO: 212) 3A 3B SE/NA EQ/GPEQ/AA EQ/AA unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 122) NO: 213)NO: 214) NO: 214) 3B 3C MQ/QP IQ/GP IQ/GG IQ/GG unknown (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 123) NO: 215) NO: 217) NO: 217) VQ/GP   (SEQ ID NO: 216) 3C 3D MQ/GL PQ/GA PQ/GA PQ/GA unknown (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 124) NO: 218) NO: 218) NO: 218) *ribosome skipping sequence

Multiple unique viruses were discovered at the USDA that are moresimilar to SVV than SVV is to other cardioviruses. These USDA virusisolates, herein considered to be members of the group called “SVV-likepicornaviruses,” are: MN 88-36695, NC 88-23626, IA 89-47552, NJ90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA99-29256; MN 99197; and SC 363649. These SVV-like picornaviruses and SVVare considered to comprise a new picornavirus genus.

Each of these SVV-like picornaviruses are unique, and are about 95%-98%identical to SVV at the nucleotide level (see FIGS. 87-89 for nucleotidesequence comparisons between SVV and these USDA isolates).

Part III: Serum Studies

Pigs are a permissive host for the USDA virus isolates identified above.The isolate MN 88-36695 was inoculated into a gnobiotic pig and antiseragenerated (GP102). The antisera binds to all of the other USDA isolateslisted above and to SVV. The antisera does not react with 24 commonporcine virus pathogens indicating its specificity. Porcine sera wasalso tested for neutralizing antibodies to 1278 (Plum Island virus).Sera were collected in the US and 8/29 sera were positive with titersranging from 1:57 to 1:36,500.

To test whether the pig is the natural source for SVV, serum samplesfrom various animals were obtained and tested for their ability to actas neutralizing antibodies against SVV infection of permissive cells.The Serum Neutralization Assay is conducted as follows: (1) Dilutevarious serums 1:2 and 1:4; (2) Mix with 100 TCID₅₀ of virus (SVV; butany virus can be tested to determine whether a serum can neutralize itsinfection); (3) Incubate at 37° C. for 1 hour; (4) Add to 1×10⁴ PER.C6cells (or other permissive cell type); (5) Incubate at 37° C. for 3days; and (6) Measure CPE using MTS assay. The neutralization titer isdefined as the highest dilution of sera that neutralizes SVV (or othervirus in question) at 100%.

The serum neutralization results showed that there is a minimal or nopresence of neutralizing antibodies in human and primate populations. Inone experiment, 0/22 human sera contained neutralizing antibodies toSVV. In another experiment, only 1/28 human sera contained neutralizingantibodies. In a third experiment, 0/50 human sera from Amish farmerswere neutralizing. In another experiment, 0/52 primate sera from fourspecies were neutralizing.

The serum neutralization results showed that there is a prevalence ofneutralizing antibodies in farm animal populations. In one experiment,27/71 porcine sera from farms were neutralizing. In another experiment,4/30 porcine sera from a disease-free farm were neutralizing. In anotherexperiment, 10/50 bovine sera were neutralizing. In yet anotherexperiment, 5/35 wild mouse sera were neutralizing. Because antibodiescross-reactive to SVV and/or SVV-like picornaviruses have been found inpigs, cows, and mice, these data indicate that SVV and/or SVV-likepicornaviruses may be prevalent in a wide-variety of non-primateanimals.

A crude viral lysate of MN 88-36695 was tested to assess itscytotoxicity ability on two cell lines permissive (NCI-H446; HEK293) forSVV and on two cell lines non-permissive (NCI-H460 and S8) for SVV. Thecytotoxicity profile for MN 88-36695 was identical to SVV: the TCID50for NCI-H446 was 1.6×10⁻⁶; the TCID50 for HEK293 was 1.3×10⁻²; andNCI-H460 and S8 were non-permissive for MN 88-36695. This data indicatesthat SVV-like picornaviruses have the potential to be used in thepresent methods directed to cancer therapy. In one embodiment, theinvention provides for the use of the MN 88-36695 SVV-like picornavirusin any of the methods directed to cancer therapy, diagnosis, orscreening.

Antisera to MN 88-36694 and SVV were tested in serum neutralizationassays on each virus. Anti-SVV mouse serum was able to neutralizeinfection by both MN 88-36695 and SVV (neutralization titers oninfection were 1:640 for MN 88-36695 and 1:1000 for SVV). Anti-MN88-36695 gnobiotic pig serum was able to neutralize infection by both MN88-36695 and SVV (neutralization titers on infection were 1:5120 for MN88-36695 and 1:100 for SVV).

These data indicate that SVV is genetically and serologically linked tothe porcine USDA virus isolates.

Example 4 SDS-PAGE and N-Terminal Sequence Analysis of SVV CapsidProteins

Purified SVV is subjected to electrophoresis using NuPAGE pre-castBis-Tris polyacrylamide mini-gel electrophoresis system (Novex, SanDiego, Calif., USA). One half of the gel is visualized by silver stainwhile the other half is used to prepare samples for amino acidsequencing of the N-termini of the capsid proteins. Prior to transfer ofproteins to membrane, the gel is soaked in 10 mM CAPS buffer, pH 11, for1 hour, and a PVDF membrane (Amersham) is wetted in methanol. Proteinsare transferred to the PVDF membrane. After transfer, proteins arevisualized by staining with Amido black for approximately 1 minute, andbands of interest are excised with a scalpel and air dried. The proteinscan be subjected to automated N-terminal sequence determination by Edmandegradation using a pulsed phase sequencer.

Three major structural proteins of the purified SVV are shown in FIG. 45(approximately 36 kDa, 31 kDa, and 27 kDa).

Example 5 Assay for Neutralization Antibodies to SVV in Human SerumSamples

Preexisting antibodies to particular viral vectors may limit the use ofsuch vectors for systemic delivery applications such as for treatment ofmetastatic cancer, because preexisting antibodies may bind tosystemically delivered vectors and neutralize them before the vectorshave a chance to transduce the targeted tissue or organ. Therefore, itis desirable to ensure that humans do not carry neutralizationantibodies to viral vectors selected for systemic delivery. To determinewhether human sera samples contain SVV-specific neutralizing antibodies,neutralization assays are carried out using randomly collected humansera samples.

Tissue culture infective dose 50: One day before the experiment, 180 μlof PER.C6 cell suspension containing 1×10⁴ cells are plated in 96-welltissue culture dish. The crude virus lysate (CVL) of SVV is diluted inlog steps from 10⁻⁰ to 10⁻¹¹ in DMEM medium (Dulbecco's Modified Eagle'sMedium) and 20 μl of each dilution is transferred to three wells of aFalcon 96-well tissue culture plate containing PER.C6 cells. The platesare incubated at 37° C. in 5% CO₂ and read at 3 days for microscopicevidence of cytopathic effect (CPE), and the tissue culture infectivedose 50 (TCID₅₀) is calculated.

Neutralization assay: First, 40 μl of medium is placed in all the wellsand then 40 μl of heat-inactivated serum is added to the first well andmixed by pipeting, making a 1:4 dilution used for screening purposes. 40μl is then transferred to the next well to perform a two-fold dilutionof the serum samples. 40 μl of SVV virus, containing 100 TCID₅₀, isadded to wells containing diluted serum samples. Plates are incubated at37° C. for 1 hour. 40 μl of the mix is taken and transferred to a platecontaining PER.C6 cells (1×10⁴ cells/160 μl/well). The plates areincubated at 37° C. for 3 days. After this time, the cultures are readmicroscopically for CPE.

In a representative neutralization assay performed as described above,twenty-two human sera samples randomly collected from USA, Europe andJapan were examined for SVV specific neutralizing antibodies. The serumsamples were serially diluted and mixed with a fixed amount of SVVcontaining 100 TCID₅₀. Serum-virus mixtures were then used to infectPER.C6 cells and incubated for 24 hours. Neutralizing antibody titer wasdetermined as the reciprocal of the highest dilution of serum able toblock CPE formation. In this experiment, no dilution of serum blockedCPE formation indicating that the human serum samples did not containSVV neutralizing antibodies.

Further SVV infection of PER.C6 was not inhibited by incubation withhuman blood (see Example 6), indicating that SVV infection was notinhibited by complement or by hemagglutination. As a result, SVVexhibits a longer circulation time in vivo than other oncolytic viruses,which is a significant problem with the use of oncolytic adenoviruses.

Example 6 Binding of SVV to Human Erythrocytes and Hemagglutination

Various viral serotypes have been shown to cause in vitrohemagglutination of erythrocytes isolated from blood of various animalspecies. Hemagglutination or binding to erythrocytes may cause toxicityin vivo and may also affect in vivo biodistribution and the efficacy ofa viral vector. Therefore, it is desirable to analyze the erythrocyteagglutination properties of a viral vector selected for systemicadministration to treat metastatic cancers.

Hemagglutination assay: To determine whether SVV causes agglutination ofhuman erythrocytes, hemagglutination assays are carried out in U-bottom96-well plates. Purified SVV is serially diluted in 25 μl PBS (PhosphateBuffered Saline) in duplicates, and an equal volume of 1% erythrocytesuspension is added to each well. Blood samples used for isolation oferythrocytes are obtained from healthy individuals with heparin as ananticoagulant. Erythrocytes are prepared by washing the blood threetimes in cold PBS to remove the plasma and the white blood cells. Afterthe last wash, erythrocytes are suspended in PBS to make a 1% (V/V) cellsuspension. The virus and erythrocytes are gently mixed and the platesare incubated at room temperature for 1 hour and monitored for ahemagglutination pattern.

Whole blood inactivation assay: To rule out direct inactivation of SVVby blood components, aliquots of virus are incubated with heparinizedhuman blood belonging to A, B, AB and O blood groups or PBS for 30minutes or 1 hour at room temperature prior to separation of plasma,after which PER.C6 cells are infected and titers are calculated.

In representative assays performed as described above, nohemagglutination of human erythrocytes of different blood groups (A, B,AB and O) was seen at any tested dilutions of SVV. A slight increase inthe virus titer is noticed when SVV is mixed with blood human samplesand incubated for 30 minutes and 1 hour, indicating that the virus isnot inactivated by blood components but becomes more infectious undertested conditions.

Example 7 In Vivo Clearance

Blood circulation time: To determine the blood circulation time and theamount of the virus in the tumor, H446 tumor bearing nude mice weretreated with SVV at a dose of 1×10¹² vp/kg by tail vein injection. Themice were bled at 0, 1, 3, 6, 24, 48, 72 hours and 7 days (189 hours)post-injection and the plasma was separated from the blood immediatelyafter collection, diluted in infection medium, and used to infect PER.C6cells. The injected mice were sacrificed at 6, 24, 48, 72 hours and 7days post-injection and the tumors were collected. The tumors were cutinto small sections and suspended in one ml of medium and subjected tothree cycles of freeze and thaw to release the virus from the infectedcells. Serial log dilutions of supernatants were made and assayed fortiter on PER.C6 cells. SVV titers were expressed as pfu/ml. The tumorsections were also subjected to H&E staining and immunohistochemistry todetect the virus capsid proteins in the tumor.

The circulating levels of virus particles in the blood were determinedbased on the assumption that 7.3% of mouse body weight is blood. Inrepresentative assays performed as essentially as described above,within 6 hours of virus administration, the circulating levels of SVVreduced to zero particles and SVV was not detectable at later timepoints (FIG. 46A). In the tumor, SVV was detectable at 6 hourspost-injection, after which the amount of the virus increased steadilyby two logs (FIG. 46B). The virus was detectable in the tumor as late as7 days postinjection (FIG. 46B). The tumor sections when subjected toimmunohistochemistry, revealed SVV proteins in the tumor cells (FIG. 47,top panels). When stained by H&E, the tumor sections revealed severalrounded tumor cells (FIG. 47, bottom panels).

SVV also exhibits a substantially longer resident time in the bloodcompared to similar doses of i.v. adenovirus. Following a single i.v.dose, SVV remains present in the blood for up to 6 hours (FIG. 46C; FIG.46C is a duplication of FIG. 46A for comparison purposes to FIG. 46D),whereas adenovirus is cleared from the blood in about an hour (FIG.46D).

Example 8 Tumor Cell Selectivity

In vitro cell killing activity of SVV: To determine the susceptibilityof human, bovine, porcine, and mouse cells, normal and tumor cells wereobtained from various sources and infected with SVV. All cell types werecultured in media and under the conditions recommended by the supplier.Primary human hepatocytes may be purchased from In Vitro Technologies(Baltimore, Md.) and cultured in Hepatocyte Culture Media (HCM™,BioWhittaker/Clonetics Inc., San Diego, Calif.).

In vitro cytopathic assay: To determine which types of cells aresusceptible to SVV infection, monolayers of proliferating normal cellsand tumor cells were infected with serial dilutions of purified SVV. Thecells were monitored for CPE and compared with uninfected cells. Threedays following infection, a MTS cytotoxic assay is performed andeffective concentration 50 (Ec₅₀) values in particles per cell arecalculated. See Tables 5 and 6 below and Table 1A supra.

TABLE 5 Cell lines with EC₅₀ values less than 100 EC50 number Cell lineswith EC50 <1 H446 (human sclc) 0.001197 PERC6 0.01996 H69AR(sclc-multidrug resistant) 0.03477 293 (human kidney transformed withad5E1) 0.03615 Y79 (human retinoblastoma) 0.0003505 IMR32 (human brain;neuroblastoma) 0.03509 D283med (human brain; cerebellum; 0.2503medulloblastoma) SK-N-AS (human brain; neuroblastoma) 0.474 N1E-115(mouse neuroblastoma) 0.002846 SK-NEP-1 (kidney, wilms' tumor, pleural0.03434 effusion, human) BEKPCB3E1 (bovine embryonic kidney cells 0.99transformed with ad5E1 Cell Lines with EC50 <10 (1-10) H1299 (human-nonsclc) 7.656 ST (pig testes) 5.929 DMS 153 (human sclc) 9.233 Cell lineswith EC50 <100 (10-100) BEK (bovine embryonic kidney) 17.55

TABLE 6 Cell lines with EC₅₀ values more than 1000 M059K (human brain;HUVEC (human vein endothelial CMT-64 (mouse-sclc) malignantglioblastoma) cells) KK (human glioblastoma) HAEC (human aorticendothelial LLC-1 (mouse-LCLC)) cells) U-118MG (human WI38 (human lungfibroblast) RM-1 (mouse-prostate) glioblastoma) DMS 79 (human sclc)MRC-5 (human lung fibroblast) RM-2 (mouse-prostate) H69 (human sclc)IMR90 (human lung fibroblast) RM-9 (mouse-prostate) DMS 114 (human sclc)HMVEC (human microvascular MLTC-1 (mouse-testes) endothelialcells-adult) DMS 53 (human sclc) HMVEC (human microvascular KLN-205(mouse-sqcc) endothelial cells-neonatal) H460 (human-LCLC) HCN-1A (humanbrain) CMT-93 (mouse-rectal) A375-S2 (human HRCE (human renal corticalB16F0 (mouse melanoma) epithelial cells) melanoma) SK-MEL-28 (humanNeuro-2A (mouse melanoma) neuroblastoma) PC3 (human prostate) C8D30(mouse brain) PC3M2AC6 (human PK15 (pig-kidney) prostate) LNCaP (humanprostate) FBRC (fetal bovine retina) DU145 (human prostate) MDBK (bovinekidney) Hep3B (human liver CSL 503 (sheep lung cells carcinoma)transformed with ad5E1) Hep2G (human liver OFRC (ovine fetal retinacarcinoma) cells) SW620 (human-colon) SW839 (human kidney) 5637 (humanbladder) HeLa S3 S8

The MTS assay was performed according to the manufacturer's instructions(CellTiter 96® AQ_(ueous) Assay by Promega, Madison, Wis.). TheCellTiter 96® AQ_(ueous) Assay preferably uses the tetrazolium compound(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS) and an electron coupling reagent, phenazinemethosulfate (PMS). Contact-inhibited normal human cells evaluated inthe study include: HUVEC (human umbilical vein endothelial cells), HAEC(human aortic endothelial cells, Clonetics/BioWhittaker #CC-2535), Wi38(normal human embryo lung fibroblasts, ATCC #CCL-75), IMR90 (humannormal lung fibroblasts, ATCC CCL-186), MRC-5 (human normal lungfibroblasts, ATCC, #CCL-171) and HRCE (human renal cortical epithelialcells, Clonetics/BioWhittaker #CC-2554).

SVV does not produce CPE in any of the above contact-inhibited normalcells. No virus-induced CPE was seen in the following human tumor celllines: Hep3B (ATCC #HB-8064), HepG2 (human hepatocellular carcinoma,ATCC #HB-8065), LNCaP (human prostate carcinoma, ATCC #CRL-10995),PC3M-2AC6, SW620 (human colorectal adenocarcinoma, ATCC #CCL-227), SW839 (human kidney adenocarcinoma, ATCC #HTB-49), 5637 (human urinarybladder carcinoma, ATCC #HTB-9), DMS-114 (small cell lung cancer, ATCC#CRL-2066), DMS 153 (human small cell lung cancer, ATCC #CRL-2064), A549(human lung carcinoma, ATCC #CCL-185), HeLa S3 (human cervicaladenocarcinoma, ATCC #CCL-2.2), NCI-H460 (human large cell lung cancer,ATCC #HTB-177), KK (glioblastoma), and U-118 MG (human glioblastoma,ATCC #HTB-15). Note—the cell lines in Table 6 with EC₅₀ values greaterthan 1000 are most likely not permissive for SVV replication and/orvirion production; although the possibility remains that SVV can bindand enter into these cells but CPE is not observed because SVVreplication cannot occur inside the cell or that replication does occurbut CPE is not observed because there is some other post-entry block(i.e., no packaging of replicated SVV genomes into virions). However,considering the absence of CPE in these cell lines, these cell-lines,and potentially tumor-types thereof, are good candidates to test whichcell and tumor-types are permissive or non-permissive for SVVreplication. Although wild-type SVV is tumor-specific, and has beenshown to target neuroendocrine tumors, including small cell lung cancerand neuroblastomas, there may be individual patients that have types ofetiologies such that SVV is not permissive in their form ofneuroendocrine tumor. Therefore, the invention does contemplate thegeneration of SVV derivatives that can kill tumor cell-types isolatedfrom individual patients where the tumors are non-permissive to thewild-type SVV, and the tumor-types isolated from these individuals caninclude, for example, glioblastoma, lymphoma, small cell lung cancer,large cell lung cancer, melanoma, prostate cancer, liver carcinoma,colon cancer, kidney cancer, colon cancer, bladder cancer, rectal cancerand squamous cell lung cancer.

SVV-mediated cytotoxicity on primary human hepatocytes (In VitroTechnologies) was determined by LDH release assay (CytoTox® 96Non-Radioactive Cytotoxicity Assay, Promega, #G1780). Primary humanhepatocytes plated in collagen coated 12-well plates were infected withSVV at 1, 10 and 100 and 1000 particles per cell (ppc). After 3 hours ofinfection, the infection medium was replaced with 2 ml of growth mediumand incubated for 3 days in a CO₂ incubator. The cell associated lactatedehydrogenase (LDH) and LDH in the culture supernatant was measuredseparately. Percent cytotoxicity is determined as a ratio of LDH unitsin supernatant over maximal cellular LDH plus supernatant LDH.

${{Percent}\mspace{14mu} {cytotoxicity}} = \frac{{LDH}\mspace{14mu} {units}\mspace{14mu} {in}\mspace{14mu} {culture}\mspace{14mu} {supernatant} \times 100}{{Sum}\mspace{14mu} {of}\mspace{14mu} {LDH}\mspace{14mu} {units}\mspace{14mu} {in}\mspace{14mu} {supernatant}\mspace{14mu} {and}\mspace{14mu} {cell}\mspace{14mu} {lysate}}$

The data shown in FIG. 48 illustrates the absence of SVV mediatedhepatoxicity at all tested multiplicity of infections.

Example 10 Virus Production Assay

To assess the replicative abilities of SVV, several selectedcontact-inhibited normal cells and actively dividing tumor cells wereinfected with SVV at one virus particle per cell (ppc). After 72 hours,cells and the medium were subjected to three freeze-thaw cycles andcentrifuged to collect the supernatant. Serial log dilutions ofsupernatants were made and assayed for titer on PER.C6 cells. For eachcell line, the efficiency of SVV replication was expressed as pfu/ml(FIG. 49).

Example 10 Toxicity

The maximum tolerated dose (MTD) is defined as the dosage immediatelypreceding the dose at which animals (e.g. mice) demonstrate a doselimiting toxicity (DLT) after the treatment with SVV. DLT is defined asthe dose at which the animals exhibit a loss in body weight, symptoms,and mortality attributed to SVV administration during the entireduration of the study. Neutralizing antibodies to SVV were assessed atbaseline, day 15, and day 21. Neutralization assays were carried asdescribed earlier.

Escalating doses (1×10⁸-1×10¹⁴ vp/kg) of SVV were administeredintravenously into both immune deficient nude and caesarean derived-1(CD-1) out-bred immune competent mice purchased from Harlan SpragueDawley (Indianapolis, Ind., USA) to determine the MTD with 10 mice perdose level. The virus was well-tolerated at all tested dose levelswithout exhibiting any clinical symptoms and without loss in body weight(FIG. 50). Mice were bled at day 15 and 21 and the sera was monitoredfor the presence of SVV-specific neutralizing antibodies inneutralization assays. SVV injected CD1 mice develop neutralizingantibodies and the titers range from 1/1024 to greater than 1/4096.

Another toxicity study was conducted on the immunocompetent mouse strain(A/J). It has been demonstrated that SVV exhibits cell killing activityand replication in N1E-115 cells (see Table 1). The murine cell lineN1E-115 (a neuroblastoma cell line, i.e., neuroendocrine cancer) isderived from the A/J mouse strain. Thus, a syngeneic mouse model wasestablished where N1E-115 cells were implanted subcutaneously in A/Jmice to form tumors, and the mice were then treated with SVV toinvestigate its efficacy and toxicity.

In the A/J study, mice were i.v. injected with SVV to determine whetherA/J mice can tolerate systemic administration of SVV. Blood hematologyresults were obtained to look for signs of toxicity, and serum chemistryresults can also be obtained. The study design is shown in Table 7below:

TABLE 7 A/J Study Design Dosage Dosage Group Animals Test Level VolumeDosing Necropsy # (Female) Article (particles/kg) (mL/kg) regimen Day 15 Vehicle  0 10 IV on Day 15 Day 1 2 5 SVV  10⁸ 10 IV on Day 15 Day 1 35 SVV  10¹¹ 10 IV on Day 15 Day 1 4 5 SVV  10¹⁴ 10 IV on Day 15 Day 1

The A/J mice were 8-10 week old females obtained from The JacksonLaboratory (Bar Harbor, Me.). SVV was prepared by storing isolatedvirions at −80° C. until use. SVV was prepared fresh by thawing on iceand diluting with HBSS (Hank's balanced salt solution). SVV was dilutedto concentrations of 10⁷ particles/mL for group 2, 10¹⁰ particles/mL forgroup 3, and 10¹³ particle/mL for group 4. HBSS was used as the vehiclecontrol for group 1. All dosing solutions were kept on wet ice untildosing.

SVV was administered to animals intravenous injection via the tail veinat a dose volume of 10 mL/kg body weight. Animals were weighed on theday of dosing and dose volumes were adjusted based on body weight (i.e.,a 0.0200 kg mouse gets 0.200 mL of dosing solution). Mice were monitoredtwice daily for morbidity and mortality. Mice were weighed twice weekly.Information relating to moribund animals and animals exhibiting anyunusual symptoms (physically or behaviorally) are recorded immediately.

Post-mortem observations and measurements entail the collection of bloodfrom all surviving animals at terminal sacrifice for standard hematologyand serum chemistry (AST, ALT, BUN, CK, LDH). The following organs areto be collected at sacrifice: brain, heart, lung, kidney, liver, andgonads. Half of each organ sample is snap frozen on dry ice and theother half will be placed in formalin.

Initial blood hematology results (CBC, differential) were obtained twoweeks after SVV injection and the results are summarized below in Table8 below. Five mice were tested from each test group (see Table 7):

TABLE 8 A/J Toxicity Results - Blood Hematology Test Group 1 Test Group2 Test Group 3 Test Group 4 Body Weight Result ± SD (g): Day 0 21.48 ±0.88  21.98 ± 1.93  22.58 ± 0.87  21.04 ± 1.67  Day 14 20.26 ± 0.93 20.92 ± 1.71  21.44 ± 0.84  21.26 ± 1.45  CBC Wet (Result ± SD (refrange)): White blood count 3.63 ± 1.57  4.5 ± 1.57 4.26 ± 0.94 4.72 ±0.62 (THSN/UL)  (2.60-10.69)  (2.60-10.69)  (2.60-10.69)  (2.60-10.69)Red blood count 9.87 ± 0.03 9.49 ± 0.07 9.76 ± 0.37 9.71 ± 0.32(MILL/UL) (6.4-9.4) (6.4-9.4) (6.4-9.4) (6.4-9.4) Hemoglobin 15.37 ±0.06  14.78 ± 0.29  15.12 ± 0.66  15.02 ± 0.63  (GM/DL) (11.5-16.1)(11.5-16.1) (11.5-16.1) (11.5-16.1) Hematocrit (%) 46.03 ± 0.40  44.52 ±0.49  45.7 ± 1.82 45.28 ± 1.69  (36.1-49.5) (36.1-49.5) (36.1-49.5)(36.1-49.5) MCV (FL) 46.67 ± 0.58  47.00 ± 0.0  47.0 ± 0.0  46.6 ± 0.55(45.4-60.3) (45.4-60.3) (45.4-60.3) (45.4-60.3) MHC (PICO GM) 15.57 ±0.06  15.70 ± 0.17 15.37 ± 0.06  15.43 ± 0.15  (14.1-19.3) (14.1-19.3)(14.1-19.3) (14.1-19.3) MCHC (%) 33.37 ± 0.12  33.14 ± 0.48  33.08 ±0.22  33.14 ± 0.25  (25.4-34.1) (25.4-34.1) (25.4-34.1) (25.4-34.1)Platelet (THSN/UL) 885.33 ± 28.6  758.2 ± 146.2 874.8 ± 56.7  897.2 ±105.4  (592-2972)  (592-2972)  (592-2972)  (592-2972) Differential(Result ± SD (ref range)): Bands (THSN/UL) 0.0 0.0 0.0 0.0 (0.0-0.1)(0.0-0.1) (0.0-0.1) (0.0-0.1) Seg. Neutrophils 0.92 ± 0.27 1.16 ± 0.371.09 ± 0.38 0.96 ± 0.20 (THSN/UL) (0.13-2.57) (0.13-2.57) (0.13-2.57)(0.13-2.57) Lymphocytes 2.64 ± 1.26 2.98 ± 1.41 3.10 ± 0.56 3.70 ± 0.41(THSN/UL) (1.43-9.94) (1.43-9.94) (1.43-9.94) (1.43-9.94) Monocytes 0.06± 0.04 0.15 ± 0.05 0.06 ± 0.03 0.05 ± 0.02 (THSN/UL)  (0.0-0.39) (0.0-0.39)  (0.0-0.39)  (0.0-0.39) Eosinophils 0.01 ± 0.01 0.01 ± 0.010.01 ± 0.01 0.003 ± 0.01  (THSN/UL)  (0.0-0.24)  (0.0-0.24)  (0.0-0.24) (0.0-0.24) Basophils 0.0 0.004 ± 0.005 0.0 0.0 (THSN/UL) (0.0-0.0)(0.0-0.0) (0.0-0.0) (0.0-0.0) Atypical Lympho. 0.0 0.0 0.0 0.0 (THSN/UL)(0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Metamyelocytes 0.0 0.0 0.0 0.0(THSN/UL) (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Myelocytes 0.0 0.0 0.00.0 (THSN/UL) (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) NRBC (/100WBC) 0.00.0 0.0 0.0 (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Other (Result ± SD(ref range)): AST (SGOT) (U/L) 1762.8 ± 1129.8 899.0 ± 234.6 779.8 ±312.2 843.2 ± 653.4  (72-288)  (72-288)  (72-288)  (72-288) ALT (SGPT)(U/L) 2171.8 ± 2792.9 535.2 ± 272.8   555 ± 350.8 380.2 ± 385.7 (24-140)  (24-140)  (24-140)  (24-140) BUN (MG/DL) 27.2 ± 0.8  24.8 ±1.9  24.6 ± 5.5  28.2 ± 12.8  (9-28)  (9-28)  (9-28)  (9-28) Creatinephospho- 28312.8 ± 20534.4 12194.4 ± 4049.2   10157 ± 5420.5   11829 ±10363.9 kinase (U/L)  (0-800)  (0-800)  (0-800)  (0-800) LDH (U/L)6650.2 ± 4788.6 3661.6 ± 933.6  3450.8 ± 972.6  2808.4 ± 1709.1(260-680) (260-680) (260-680) (260-680) Hemolytic Index 706.6 ± 423.4477.6 ± 195.7 589.6 ± 198.6 496.4 ± 321.1 (MG/DL HGB)  (0-70)  (0-70) (0-70)  (0-70)

These results show that there are no abnormalities in blood hematologyprofiles obtained from mice treated with low, medium and high doses ofSVV compared to blood hematology profiles obtained from untreated mice.From this study, it can be concluded that there are no measureable signsof toxicity following systemic administration of SVV, indicating thatSVV is tolerated by A/J mice following i.v. injection.

Example 11 Efficacy

Athymic female nude mice (nu/nu) aged 6-7 weeks purchased from HarlanSprague Dawley (Indianapolis, Ind.) were used in efficacy studies. Micewere injected subcutaneously with 5×10⁶ H446 cells into the right flankusing manual restraint. Tumor sizes were measured regularly, and thevolumes were calculated using the formula π/6×W×L², where L=length andW=width of the tumor. When the tumors reach approximately 100-150 mm³,mice (n=10) were randomly divided into groups. Mice were injected withescalating doses of SVV by tail vein injections at a dose volume of 10ml/kg. A control group of mice was injected with an equivalent volume ofHBSS. Dose escalation proceeds from 1×10⁷ to 1×10¹³ particles perkilogram body weight. Antitumoral efficacy was determined by measuringtumor volumes twice weekly following SVV administration. Completeresponse was defined as complete disappearance of xenograft; partialresponse as regression of the tumor volume by equal to or more than 50%;and no response as continuous growth of tumor as in the control group.

Tumors from mice treated with HBSS grew rapidly and the tumor volumesreached more than 2000 mm³ by study day 20 (FIG. 51; see line with opendiamond). In contrast, mice given one systemic injection of SVV at alltested doses (with the exception of the lowest dose) became tumor freeby study day 20. In the lowest dose group, 8 mice became tumor free, onemouse had a very large tumor and the other had a small palpable tumor(25 mm³) by study day 31. To evaluate the antitumor activity of SVV onlarge sized tumors, five mice from HBSS group bearing tumors >2000 mm³were systemically injected with a single dose of 1×10¹¹ vp/kg on studyday 20. For the duration of the follow-up period (11 days of after SVVinjection), a dramatic regression of the tumor volumes were noted (FIG.51).

Additional experiments to test the efficacy of a single intravenous doseof SVV was conducted in murine tumor models that express neuroendocrinemarkers. The tumor models tested included H446 (human SCLC) (see FIG.90A), Y79 (human retinoblastoma) (see FIG. 90B), H69AR (human multi-drugresistant SCLC) (see FIG. 90C), H1299 (human NSCLC) (see FIG. 90D), andN1E-115 (murine neuroblastoma) (see FIG. 90E).

The results show that a single intravenous dose of SVV has efficacy inall of the murine neuroendocrine tumor models. The results also showthat SVV is efficacious in the N1E-115 immunocompetent murineneuroblastoma model.

FIG. 52 shows a picture of mice that were “untreated” with SVV (i.e.,treated with HBSS) or “treated” with SVV. As can be seen, the untreatedmice had very large tumors and the treated mice showed no visible signsof tumor. Further, for unsacrificed mice treated with SVV, no tumorregrowth was observed for the duration of the study, 200 days.

In vitro efficacy data for SVV for specific tumor cell lines is shown inTables 1, 1A, and 5. The data shows that SVV specifically infectsparticular tumor cell types and does not infect normal adult cells(except for porcine normal cells), a significant advantage over anyother known oncolytic virus. SVV has been shown to have 1,000 timesbetter cell killing specificity than chemotherapy treatments (cellkilling specificity values for SVV have been shown to be greater than10,000, whereas cell killing specificity values for chemotherapy arearound 10).

Specific cytotoxic activity of SVV was demonstrated in H446 human SCLCcells. Following a two-day incubation with increasing concentrations ofSVV, cell viability was determined. The results are shown in FIG. 53.FIG. 53 shows cell survival following incubation of SVV with either H446SCLC tumor cells (top graph) or normal human H460 cells (bottom graph).SVV specifically killed the tumor cells with an EC₅₀ of approximately10⁻³ particles per cell. In contrast, normal human cells were not killedat any concentration of SVV. Further, as summarized in Tables 1, 1A-3,SVV was also cytotoxic toward a number of other tumor cell lines,including SCLC-multidrug resistant tumor cells, and some fetal cells andcell lines. The EC₅₀ values for SVV cytotoxicity for the other tumorcell lines ranged from 10⁻³ to greater than 20,000 particles per cell.SVV was non-cytotoxic against a variety other non-neural tumors andnormal human tissues. Additionally, SVV was not cytotoxic to primaryhuman hepatocytes, as measured by LDH release at up to 1000 particlesper cell (see FIG. 48).

Example 12 Biodistribution and Pharmacokinetic Study in Rodents

Pharmacokinetic and biodistribution study of SVV is performed in normalmice and immunocompromised athymic nude mice bearing H446 SCLC tumors.This study evaluates the biodistribution, elimination and persistence ofSVV following a single intravenous administration to both normal andimmunocompromised tumor-bearing mice. Groups of mice each receive asingle i.v. dose of control buffer or one of three doses of SVV (10⁸,10¹⁰, or 10¹² vp/kg) and are monitored for clinical signs. Blood samplesare obtained from groups of 5 mice at 1, 6, 24 and 48 hours post dose,and at 1, 2, 4, and 12 weeks post dose. Dose levels include a known lowefficacious dose and two higher dose levels to determine linearity ofvirus elimination. Groups of mice are sacrificed at 24 hours, and 2, 4and 12 weeks post dose. Selected tissues, including liver, heart, lung,spleen, kidney, lymph nodes, bone marrow, brain and spinal cord tissuesare aseptically collected and tested for the presence of SVV RNA using avalidated RT-PCR assay.

Samples of urine and feces are obtained at sacrifice, at 24 hours, andat 2, 4 and 12 weeks post dose and are examined for the presence ofinfectious virus. The design of the experiments in this Example areshown in Table 9 below:

TABLE 9 Biodistribution of SVV in CD-1 Mice and Athymic Nude MiceBearing SCLC Tumors # of # of Mice/ Dose Mice/Timepoint Timepoint Levelfor Blood for PCR Tissue Group Treatment (vp/kg) Route SamplingDistribution Normal CD-1 Mice 1 Saline 0 i.v. 5 5 2 SVV 10⁸  i.v. 5 5 3SVV 10¹⁰ i.v. 5 5 4 SVV 10¹² i.v. 5 5 Athymic Tumor Bearing Mice 5Saline 0 i.v. 5 5 6 SVV 10⁸  i.v. 5 5 7 SVV 10¹⁰ i.v. 5 5 8 SVV 10¹²i.v. 5 5

Acute i.v. toxicology studies were also performed in both normal andimmunocompromised athymic nude mice bearing H446 SCLC tumors.Preliminary i.v. studies in normal and SCLC tumor bearing mice indicatesafety of SVV at doses up to 10¹⁴ vp/kg. No adverse clinical signs wereobserved and there was no loss of body weight up to 2 weeks following asingle i.v. dose of 10¹⁴ vp/kg.

Example 13 Viral Transmission Study in Normal Adult and Pregnant Mice

The purpose of this Example is to determine if SVV is transmissiblefollowing cohabitation of noninfected normal mice with mice injectedwith a high concentration of SVV. Because SVV does not replicate innormal, non-tumor bearing mice, tumor bearing mice can also be injectedwith high concentrations of SVV and subsequently exposed to normal,healthy animals to better simulate the clinical scenario. A secondarypurpose is to assess the potential transmissibility of SVV from aninfected female to an uninfected pregnant DAM, and subsequently to thedeveloping fetus.

Three groups of five naive male and female CD-1 mice are exposed to asingle mouse of the same sex infected with either 10⁸, 10¹⁰ or 10¹²/kg,and are monitored for the presence of SVV by blood sampling.

Similarly, an SVV exposed female is co-mingled with a number of timedpregnant females, and the ability of the virus to transmit from theinfected female to an uninfected pregnant female, and subsequently tothe developing fetus is determined

Example 14 Non-Human Primate Studies

The safety, toxicity and toxicokinetics of SVV are also determined innon-human primates. In a dose range-finding phase, individual monkeysreceive a single i.v. dose of SVV at 10⁸ vp/kg and are closely monitoredfor clinical signs of infection or toxicity. If this dose is welltolerated, additional animals are treated with a higher i.v. dose untila dose of 10¹² vp/kg is achieved. Subsequently, the main study consistsof groups of three male and female monkeys, and each monkey is dosedonce weekly for six weeks with either vehicle alone or one of threedoses of SVV and monitored for signs of toxicity. An additional twomonkeys per sex are dosed with the vehicle alone and with the high doselevel of SVV for six weeks, and are allowed an additional four weeksrecovery prior to sacrifice.

Blood samples are obtained following dosing during week 1 and week 6.Clinical pathological and hematology blood samples are obtained prior tothe initial dose and prior to sacrifice. Additional blood samples areobtained following each dose for assessing the presence of neutralizingantibodies to SVV.

Surviving monkeys are euthanized and subjected to a full gross necropsyand a full tissue list is collected from the main study and recoverymonkeys. Tissues from the control and high dose groups are evaluatedhistopathologically. Urine and fecal samples are collected followingdosing on weeks 1 and 6 and are evaluated for presence of infectiousSVV. The overall design of this Example is shown in Table 10 below.

TABLE 10 Multiple Dose Toxicology Study of SVV in Primates DoseRange-finding Phase Group Treatment Dose (vp/kg) Route Males Females 1SVV 10⁸   IV 1 1 2 SVV 10¹⁰* IV 1 1 3 SVV 10¹²* IV 1 1 Main Phase DoseMain Phase Recovery Group Treatment (vp/kg) Route Male Female MaleFemale 1 Control — IV 3 3 2 2 2 SVV 10⁸*  IV 3 3 — — 3 SVV 10¹⁰* IV 3 3— — 4 SVV 10¹²* IV 3 3 2 2 *Doses can vary based on results of DoseRange-finding phase

Example 15 Construction of an Infectious Full-Length and FunctionalGenomic SVV Plasmid

With SEQ ID NO:1, only about 1.5-2 Kb of the 5′ genomic sequence of SVVremains to be sequenced, representing the nucleotide region covering the5′ UTR, 1A (VP4) and part of 1B (VP2). To clone the 5′ end missing inSEQ ID NO:1, polymerases that function at high temperatures and reagentsthat can enable a polymerase to read through secondary structures wereused. Additional SVV cDNAs were prepared from isolated SVV of ATCCdeposit number PTA-5343. SVV particles were infected into a permissivecell line, such as PER.C6, and viruses are isolated. Viral RNA was thenrecovered from the virus particles such that cDNA copies are madetherefrom. Individual cDNA clones were sequenced, such that selectedcDNA clones are combined into one full-length clone in a plasmid havinga T7 promoter upstream of the 5′ end of the SVV sequence. Thefull-length genomic sequence of SVV is listed in FIGS. 83A-83H and SEQID NO:168. The full-length SVV from this plasmid is reverse-transcribed,by utilizing T7 polymerase and an in vitro transcription system, inorder to generate full-length RNA (see FIG. 55). The full-length RNA isthen transfected into permissive cell lines to test the infectivity ofthe full-length clone (see FIG. 55).

The methodology was as follows.

RNA Isolation:

SVV genomic RNA was extracted using guanidium thiocyanate and a phenolextraction method using Trizol (Invitrogen). Briefly, 250 μl of thepurified SVV (˜3×10¹² virus particles) was mixed with 3 volumes ofTrizol and 240 μl of chloroform. The aqueous phase containing RNA wasprecipitated with 600 μl isopropanol. The RNA pellet was washed twicewith 70% ethanol, dried and dissolved in sterile DEPC-treated water. Thequantity of RNA extracted can be estimated by optical densitymeasurements at 260 nm. An aliquot of RNA can be resolved through a1.25% denaturing agarose gel (Cambrex Bio Sciences Rockland Inc.,Rockland, Me. USA) and the band visualized by ethidium bromide stainingand photographed.

cDNA Synthesis:

cDNA of the SVV genome was synthesized by RT-PCR. Synthesis of cDNA wasperformed under standard conditions using 1 μg of RNA, AMV reversetranscriptase, and oligo-dT primers. Random 14-mer oligonucleotide canalso be used. Fragments of the cDNA were amplified and cloned into theplasmid pGEM-3Z (Promega) and the clones were sequenced. The sequence atthe 5′ end of the viral genome was cloned by RACE and the sequencedetermined Sequence data was compiled to generate the complete genomesequence of SVV.

Cloning of Full Length Genome:

Three cDNA fragments representing the full-length SVV genome wereamplified by three PCR reactions employing six sets of SVV-specificprimers. Turbo pfu polymerase (Stratagene) was used in PCR reactions.First, a fragment representing the 5′ end of SVV genome was amplifiedwith primers 5′SVV-A (SEQ ID NO:219) and SVV1029RT-R1 (SEQ ID NO:220)and the resulting fragment was cut with ApaI and EcoRI and gel purified.The gel purified fragment was ligated to Nde-ApaT7SVV (SEQ ID NO:221),an annealed oligo duplex containing engineered NdeI site at 5′ end, T7core promoter sequence in the middle and first 17 nucleotides of SVVwith ready to use ApaI site at 3′ end and cloned into Nde I and Eco RIsites of pGEM-3Z (Promega) by three-way ligation to generate pNTX-03.Second, a fragment representing 3′ end of viral genome was amplified byPCR with primers SVV6056 (SEQ ID NO:222) and SVV7309NsiB (SEQ IDNO:223). The antisense primer, SVV7309NsiB was used to introducedpoly(A) tail of 30 nucleotides in length and Nsi I recognition sequenceat 3′ end to clone into PstI site of pGEM-3Z plasmid. The resulting PCRproduct was digested with BamHI and gel purified. A fragment coveringthe internal part of the viral genome was amplified with primers SVV911L(SEQ ID NO:224) and SVV6157R (SEQ ID NO:225). The resulting PCR productwas cut with EcoRI and BamHI and gel purified. The two gel purifiedfragments representing the middle and 3′ end of SVV genome were clonedinto EcoRI and SmaI sites of pGEM-4Z by three-way ligation to generatepNTX-02. To generate full-length SVV cDNA, pNTX-02 was digested withEcoRI and NsiI and the resulting 6.3 kb fragment was gel purified clonedinto EcoRI and PstI sites of pNTX-03. The resulting full-length plasmidwas called pNTX-04.

The full-length plasmid pNTX-04 was further modified at both 5′ and 3′ends to facilitate in vitro transcription and rescuing of the virusfollowing RNA transfection into PER.C6 cells. First, a SwaI restrictionenzyme site was inserted immediately downstream of the poly(A) tail toliberate the 3′ end of SVV-cDNA from the plasmid backbone prior to invitro transcription. A PCR approach was used to insert the siteutilizing a primer pair of SVV6056 (SEQ ID NO:222) and SVVSwaRev (SEQ IDNO:226) and pNTX-04 as template. The antisense primer SVV3SwaRevcontained 58 nucleotides representing the 3′ end of the SVV sequence andrecognition sequences for SwaI and SphI restriction enzyme sites. Theresulting PCR fragment was digested with BamHI and SphI and used toreplace the corresponding fragment from pNTX-04 to generate pNTX-06.Second, an extra four nucleotides present between the T7 promotertranscription start site and 5′ end of SVV cDNA in pNTX-06 were removedusing annealed oligo duplex approach. The duplex nucleotides wereengineered to contain KpnI recognition site, T7 core promoter sequenceand the first 17 nucleotides of SVV with a ready to use ApaI site at the3′ end (SEQ ID NO:227). The annealed oligos were used to replace thecorresponding portion of pNTX-06 using KpnI and ApaI sites to generatepNTX-07. Finally, a two base pair deletion noticed in the polymeraseencoding region of pNTX-07 was restored by replacing BamHI and SphIfragment with a corresponding fragment amplified from SVV cDNA by PCR togenerate pNTX-09.

In Vitro Transcription:

Infectivity of in vitro transcribed RNA was tested by first digestingpNTX-09 with SwaI to liberate 3′ end of SVV sequence from plasmidbackbone. The linearized plasmid was subjected to in vitro transcriptionusing T7 polymerase (Promega).

Transfection of In Vitro Transcribed RNA into PER.C6 Cells:

One day prior to transfection, PER.C6 cells were plated in 6-welltissue-culture dishes. On the next day, Lipofetamine reagent(Invitrogen) was used to transfect in vitro transcribed RNA (1.5 ng)into the cells following the recommendations of the supplier. Cytopathiceffect (CPE) due to virus production was noticed within 36 hourpost-transfection. The transfected cells were subjected to three cyclesof freeze-thaw and the viruses in lysate were further confirmed byinfecting PER.C6 cells. Thus, the full-length SVV cDNA clone proved tobe infectious.

As described above, the plasmid with the full-length genome of SVV canbe reverse-transcribed following standard protocols. The viral RNA (100ng) can be used to transfect any cell line known to be permissive forthe native SVV, but the most efficient cell line for viral RNAtransfection can be empirically determined among a variety of celllines.

Example 16 Construction of an RGD-Displaying SVV Library

To find the optimal insertion position for the construction of SVVcapsid mutants generated with random with oligonucleotides encodingrandom peptide sequences, a simple model system (RGD) is employed. RGD(arginine, glycine, aspartic acid) is a short peptide ligand that bindsto integrins. A successful RGD-SVV derivative should contain thefollowing characteristics: (1) the genetic insertion should not alterany of SVV's unique and desirable properties; and (2) a successful RGDderivative virus should have tropism toward α_(V)β₅ integrin containingcells.

A SVV plasmid containing just the contiguous capsid region will besingly cleaved at random positions and a short model peptide sequence,referred to as RGD, will be inserted at each position. The virus SVV-RGDlibrary will be constructed from this plasmid library utilizing thegeneral technology described in FIGS. 56 and 57.

Random insertion of the cRGD oligonucleotide into the capsid region isconducted. In brief, a plasmid is constructed that just encodes thecontiguous 2.1 Kb capsid region of SVV (see FIG. 56, “pSVVcapsid”). Asingle random cleavage is made in pSVVcapsid by partially digesting theplasmid utilizing either CviJI or an endonuclease V method as describedbelow (see FIG. 57). After isolating the single cleaved plasmid the RGDoligonucleotide will be inserted to create a pSVVcapsid-RGD library.

The restriction enzyme CviJI has several advantages over other randomcleavage methods such as sonication or shearing. First, as CviJI is ablunt ended cutter no repair is necessary. Second, CviJI has beendemonstrated to cleave at random locations such that no hot spots willoccur. The procedure is also simple and rapid. Briefly, theconcentration of CviJI and/or time of digestion are increasingly lowereduntil the majority of cleaved DNA is a linearized plasmid, i.e. a singlecleavage. This can be observed by standard agarose gel electrophoresisas depicted in FIG. 57. The band is then isolated, purified and ligatedwith the RGD oligo.

Another method that may be utilized to randomly cleave DNA is theendonuclease V method (Kiyazaki, K., Nucleic Acids Res., 2002, 30(24):e139). Endonuclease V nicks uracil-containing DNA at the second or thirdphosphodiester bond 3′ to uracil sites. This method is also expected torandomly cleave DNA, the frequency is simply determined by adjusting theconcentration of dUTP in the polymerase chain reaction. Although thecleavage sites are always two or three bases downstream of a thymidine(substituted by uracil) site, this method is expected to produce muchfewer hot and cold spots than other methodologies.

The randomly linearized plasmids are ligated with the cRGDoligonucleotides. The resultant pSVV capsid library is then amplified,such that a population of polynucleotides encoding the capsid regionwith randomly inserted cRGD regions can be purified (see FIGS. 57 and58). The population of capsid polynucleotides is then subcloned into avector containing the full-length SVV sequence minus the capsid region,such that a library of full-length SVV sequences are generated (wherethe library manifests sequence diversity in the capsid region as thecRGD sequence is randomly inserted). This library is then reversetranscribed into RNA, and the RNA is transfected into a permissive cellline to generate a population of SVV particles having different capsids(see FIG. 59). Once this RGD-SVV population of virus particles isrecovered (“RGD-SVV library”), a number of viruses (i.e., 10 or more)will be randomly picked for sequencing to confirm the insertion of theRGD sequence and diversity of insertion site.

In Vitro Selection of the RGD-Displaying SVV Library.

The SVV-RGD library is screened to determine which insertion siteenabled an expanded tropism of SVV. The RGD-SVV library is allowed toinfect α_(V)β₅ integrin-expressing NSCLC lines (non-small cell lungcancer cell lines, i.e., A549 expressing α_(V)β₅). Only those SVVderivatives that contain a functional and properly displayed RGD motifcan infect these cells and replicate.

In vitro screening is carried out by a high throughput automation system(TECAN) that is capable of liquid handling, concurrent incubation of 20cell lines and measurement in 384-well plates (see FIG. 62 and FIG. 63).The cells are harvested 30 hr after infection when complete CPE isnoticed and then cells are collected by centrifugation at 1500 rpm for10 minutes at 4° C. The cell pellets are then resuspended in the cellculture supernatant and subjected to three cycles of freeze and thaw.The resulting suspension is clarified by centrifugation at 1500 rpm for10 minutes at 4° C. Virus is purified by two rounds of CsCl gradients: aone-step gradient (density of CsCl 1.24 g/ml and 1.4 g/ml) followed byone continuous gradient centrifugation (density of CsCl 1.33 g/ml). Thepurified virus concentration is determined spectrophotometrically,assuming 1A₂₆₀=9.5×10¹² particles (Scraba, D. G. and Palmenberg, A. C.,1999). The process may be repeated multiple times until a sufficientamount of virus is recovered from α_(V)β₅ cells.

Analysis of Recovered RGD-SVV Derivatives.

A small pool of individual RGD-displaying SVV derivatives (about 10-50different derivatives) are analyzed. The viral mixture is diluted andsingle viral particles are expanded for analysis. Each derivative istested to determine whether they have gained the ability to infectα_(V)β₅-expressing cells efficiently and specifically. The capsid regionof each derivative with this property is then be sequenced to determinethe site of RGD insertion. The recovered cRGD-displaying SVV derivativesshould possess the following properties: (1) the original properties ofthe virus are still intact; and (2) the derivatives have gained theability to infect cells that express high levels of integrins that bindto RGD. This approach aims to identify one or more sites that enable anexpanded tropism with RGD insertion, such that random oligonucleotidescan be inserted into these sites to generate SVV derivatives withaltered tropism.

The sequenced cRGD-SVV derivatives are numbered and ranked by theirbinding abilities to integrin. To test the binding activity, recombinantβ₂ integrin is immobilized on a 96-well microtiter plate in PBS, washedtwice with PBS, blocked with 3% BSA in PBS, and then incubated with aunique RGD-displaying virus. The native virus without peptide insertionsis used as a negative control. After 1-5 hr of incubation, the wells arewashed at least three times with PBS. Then, the viruses that are boundto the plate will be detected by anti-SVV antibodies. RGD peptide orantibodies against integrin should be able to compete with the bindingof the RGD-SVV derivatives to the integrin-bound plate.

The cRGD-SVV derivatives (20) that have the strongest binding tointegrin are analyzed to determine the ‘successful’ location(s) of cRGDoligonucleotide insertion. The insertion sites provide insights into thetropism of SVV. Based on the analysis of the insertion sites and otherknown structures, an ideal location to place a random peptide librarycan be determined (this method is an alternative method for generatingSVV derivatives, where oligonucleotides (known sequence or randomsequence) are inserted into random locations in the capsid). SVVderivatives generated with random sequence oligonucleotides areconstructed in essentially the same manner as described above for theRGD-SVV library, except for two additional and novel methodologies. Toavoid unwanted stop codons and deleterious amino acid insertions (e.g.cysteines or prolines) within a desired coding region, TRIM(trinucleotide-mutagenesis) technology developed by Morphosys (Munich,Germany) can be used to generate random oligonucleotides for capsidinsertion. TRIM utilizes tri-nucleotides which only code for amino acidsat the desired position (Virnekas, B. et al., Nucleic Acids Res, 1994,22(25): 5600-5607). The random-peptide displaying SVV with a diversityof 10⁸ is believed to be sufficient and expected to yield peptides thatspecifically direct the virus to targeted tumor tissues. Random-peptidedisplaying SVV is tested in vitro as described above, or in vivo usingtumor-bearing mice.

Example 17 Serum Studies

Pigs are a permissive host for the USDA virus isolates identified above.The isolate MN 88-36695 was inoculated into a gnotobiotic pig andantisera generated (GP102). The antisera binds to all of the other USDAisolates listed above and to SVV. The antisera does not react with 24common porcine virus pathogens indicating its specificity. Porcine serawas also tested for neutralizing antibodies to 1278 (Plum Island virus).Sera were collected in the US and 8/29 sera were positive with titersranging from 1:57 to 1:36,500.

To test whether the pig is the natural source for SVV, serum samplesfrom various animals were obtained and tested for their ability to actas neutralizing antibodies against SVV infection of permissive cells.The Serum Neutralization Assay is conducted as follows: (1) Dilutevarious serums 1:2 and 1:4 and serially in increasing dilutions ifnecessary; (2) Mix with 100 TCID₅₀ of virus (SVV; but any virus can betested to determine whether a serum can neutralize its infection); (3)Incubate at 37° C. for 1 hour; (4) Add the mixture to 1×10⁴ PER.C6®cells (or other permissive cell type); (5) Incubate at 37° C. for 3days; and (6) Measure CPE using a tetrazolium based dye cytotoxicity(such as MTS) assay. The neutralization titer is defined as the highestdilution of sera that neutralizes SVV (or other virus in question) at100%.

The serum neutralization results showed that there is a minimal or nopresence of neutralizing antibodies in human and primate populations. Inone experiment, 0/22 human sera contained neutralizing antibodies toSVV. In another experiment, only 1/28 human sera contained neutralizingantibodies. In a third experiment, 0/50 human sera from Amish farmerswere neutralizing. In another experiment, 0/52 primate sera from fourspecies were neutralizing.

The serum neutralization results showed that there is a prevalence ofSVV neutralizing antibodies in farm animal populations. In oneexperiment, 27/71 porcine sera from farms were neutralizing. In anotherexperiment, 4/30 porcine sera from a disease-free farm wereneutralizing. In another experiment, 10/50 bovine sera wereneutralizing. In yet another experiment, 5/35 wild mouse sera wereneutralizing.

Antisera to MN 88-36694 were tested in serum neutralization assays onSVV (see Example 2). Anti-MN 88-36695 gnotobiotic pig serum was able toneutralize infection by SVV (neutralization titer on infection was 1:100for SVV). As stated above, the antisera binds to all of the other USDAisolates and to SVV, indicating that the herein disclosed USDA isolatesare SVV-like picornaviruses due to their serological cross-reactivitywith the gnotobiotic pig serum as measured in an indirectimmunofluorescence assay.

These data indicate that SVV is genetically and serologically linked tothe porcine USDA virus isolates.

Example 18 SVV and SVV-Like Picornaviruses

The grouping of the following isolates: MN 88-36695, NC 88-23626, IA89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL94-9356; MN/GA 99-29256; MN 99197; and SC 363649, was deduced in partfrom indirect immunofluorescence experiments. Antisera GP102 was raisedagainst isolate MN 88-36695 by inoculation of the virus into agnotobiotic pig. The antisera binds to all twelve isolates demonstratingthat they are serologically related to one another.

The GP102 antisera was tested in a neutralization assay with SVV. Inthis assay, serial dilutions of antisera are mixed with a known quantityof SVV (100 TCID₅₀). The mixtures are placed at 37° C. for 1 hour. Analiquot of the mixture is then added to 1×10⁴ PER.C6® cells, or anothercell line that is also permissive for SVV, and the mixtures are placedat 37° C. for 3 days. The wells are then checked for a cytopathic effectof the virus (CPE). If the serum contains neutralizing antibodies, itwould neutralize the virus and inhibit the infection of the PER.C6®cells by the virus. CPE is measured quantitatively by using atetrazolium based dye reagent that changes absorbance based on thenumber of live cells present. The results are expressed as the percentof viable cells of an uninfected control vs. the log dilution of serum,and are shown in FIG. 93. This data indicates that SVV is serologicallylinked to the porcine USDA virus isolates.

Additionally, the viral lysate of MN 88-36695 was tested in cytotoxicityassays with four different cell lines and the results are shown in Table4. The permissivity profile is identical to that of SVV in that NCI-H446and HEK293 are permissive for SVV, and NCI-H460 and S8 are not.Additionally, MN 88-36695, like SVV, was cytotoxic to PER.C6® cells.Further, polyclonal antisera to SVV raised in mice was used in aneutralization assay along with MN 88-36695 virus. The results are shownin FIG. 94. The anti-SVV antisera neutralized MN 88-36695, furtherlinking SVV to the USDA viruses serologically.

TABLE 11 MN 88-36695 Cytotoxicity Results Cell Line TCID50 (pfu/ml)Result NCI-H446 1.6 × 10−6 Permissive HEK293 1.3 × 10−2 PermissiveNCI-H460 0 Nonpermissive S8 0 Nonpermissive

Partial genomic sequence analysis of several of the USDA isolatesrevealed that they are all very closely related to SVV (see FIGS. 87-89for sequence alignments). Table 12 shows the percent sequence identitybetween SVV and six of the isolates. It was found that about 95-98%identity exists at the nucleotide (nt) level over 460 nt of the 3′ endof the genome encoding 3D^(pol) and the 3′UTR (FIG. 89). Each of theUSDA viruses is unique and is about 95-98% identical to SVV at thenucleotide level.

TABLE 12 Percent Sequence Identity Between SVV and Six USDA Isolates 1 23 4 5 6 7 Virus Name 96.5 99.1 97.2 97.0 97.4 97.0 1 NJ 90-10324 97.095.7 94.8 95.0  98.3* 2 CA 13195 97.6 97.2 97.6 97.2 3 IA 89-47752 95.496.1 96.3 4 IL 92-48963 98.9 95.2 5 MN 88-36695 95.4 6 NC 88-23626 7SVV-001 (SVV)

Further sequencing of parts of the P1 (FIG. 87) and 2C (FIG. 88) genesof two of the isolates has confirmed this close relationship with SVV.The USDA isolates are more highly related to SVV than any other knownviruses, including members of the genus Cardiovirus. Sequences fromseveral regions of seven of the USDA viruses were compared with SVV andneighbor-joining trees were constructed (FIGS. 95A and 95B). These treesfurther confirm the high degree of relation between the viruses, andidentifying CA 131395 as SVV's current closest relative.

What is claimed:
 1. An isolated nucleic acid comprising a nucleic acidsequence having at least 75% sequence identity to: (i) SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, or 168, or (ii) a contiguous portion ofSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 168, that is atleast 20 nucleotides in length.
 2. The isolated nucleic acid of claim 1,wherein the nucleic acid is RNA or DNA.
 3. An isolated polypeptidecomprising an amino acid sequence having at least 75% sequence identityto: (i) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 169, or(ii) a contiguous portion of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, or 169 that is at least 10 amino acids in length.
 4. An isolatedSeneca Valley virus or derivative thereof, comprising identifyingcharacteristics of ATCC Patent Deposit number PTA-5343.
 5. An isolatedSeneca Valley virus or derivative or relative thereof, having a genomecomprising a sequence that is at least 95%, 90%, 85%, 80%, 75%, 70%, or65% identical to SEQ ID NO:1 or SEQ ID NO:168.
 6. The virus of claim 5comprising the following characteristics: replication competence intumor cells, tumor-cell tropism, and lack of cytolysis in normal cells.7. The virus of claim 6, wherein said virus is replication competent intumor cell types having neuroendocrine properties.
 8. A pharmaceuticalcomposition comprising an effective amount of the virus of claim 5 and apharmaceutically acceptable carrier.
 9. An isolated antibody thatspecifically binds to the epitope of the isolated virus of claim
 5. 10.A method for treating cancer comprising administering an effectiveamount of a virus or derivative thereof, so as to treat the cancer,wherein the virus has a genome that comprises a sequence that is atleast 75% identical to a contiguous sequence of SEQ ID NO:1 or SEQ IDNO:168 that is at least 100 nucleotides in length.
 11. The method ofclaim 10, wherein the virus is a picornavirus.
 12. The method of claim11, wherein the picornavirus is a cardiovirus.
 13. The method of claim12, wherein the cardiovirus is selected from the group consisting of:vilyuisk human encephalomyelitis virus, Theiler's murineencephalomyelitis virus, and encephalomyocarditis virus.
 14. The methodof claim 11, wherein the picornavirus is a member of a genus to whichSeneca Valley virus belongs.
 15. The method of claim 11, wherein thepicornavirus is Seneca Valley virus.
 16. The method of claim 15, whereinthe Seneca Valley virus has an ATCC deposit number PTA-5343.
 17. Themethod of claim 11, wherein the picornavirus is a Seneca Valleyvirus-like picornavirus.
 18. The method of claim 17, wherein the SenecaValley virus-like picornavirus is selected from the group of isolatesconsisting of: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL92-48963, CA 131395, LA 1278, IL 66289, IL 94-9356, MN/GA 99-29256, MN99197, and SC
 363649. 19. A method of killing an abnormallyproliferative cell comprising contacting the cell with the virus ofclaim
 5. 20. A method for making an oncolytic virus, the methodcomprising: (a) comparing a Seneca Valley virus genomic sequence with atest virus genomic sequence; (b) identifying at least a first amino aciddifference between a polypeptide encoded by the Seneca Valley virusgenomic sequence and a polypeptide encoded by the test virus genomicsequence; (c) mutating the test virus genomic sequence such that thepolypeptide encoded by the test virus genomic sequence has at least oneless amino acid difference to the polypeptide encoded by the SenecaValley virus genomic sequence; (d) transfecting the mutated test virusgenomic sequence into a tumor cell; and (e) determining whether thetumor cell is lytically infected by the mutated test virus genomicsequence.
 21. The method of claim 20, wherein the Seneca Valley virusgenome comprises a sequence that is at least 95% identical to SEQ IDNO:1 or SEQ ID NO:168.
 22. The method of claim 20, wherein the testvirus is a picornavirus.
 23. The method of claim 22, wherein the testvirus is a Seneca Valley virus-like picornavirus.
 24. The method ofclaim 20, wherein the amino acid differences are between a Seneca Valleyvirus capsid protein and a test virus capsid protein.
 25. The method ofclaim 20, wherein mutating the test virus genomic sequence comprisesmutating a cDNA having the test virus genomic sequence.
 26. The methodof claim 20, wherein transfecting the mutated test virus genomicsequence comprises transfecting RNA, wherein the RNA is generated fromthe cDNA having the mutated test virus genomic sequence.